Compositions and methods for alphavirus vaccination

ABSTRACT

Compositions of a recombinant adenovirus based vector vaccine containing one or more alphavirus antigen genes are disclosed herein. Methods for constructing and producing such vaccines and methods of using these vaccines to generate broad based immune responses against alphaviruses are also described. Compositions described herein allow for vaccinations in individuals with preexisting immunity to adenovirus.

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Patent Application No. 62/363,136 filed Jul. 15, 2016, the disclosure of which is herein incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 7, 2017, is named 39891-725_601_SL.txt and is 460,196 bytes in size.

BACKGROUND

Vaccines help the body fight disease by training the immune system to recognize and destroy harmful substances and diseased cells.

Viral vaccines are currently being developed to prevent infectious diseases and treat existing cancers. These viral vaccines work by inducing expression of a small fraction of genes or complete genes associated with a disease within the host's cells, which in turn, enhance the host's immune system to identify and destroy diseased cells. As such, clinical response due to a viral vaccine can depend on the ability of the vaccine to induce a high-level immunogenicity and have sustained long-term expression.

Therefore, there remains a need to discover novel compositions and methods for enhanced protective or cancer therapeutic responses to complex diseases and especially for newly emerging disease threats.

SUMMARY

In various aspects, the present disclosure provides composition comprising a replication defective virus vector comprising a sequence encoding an alphavirus target antigen. In some aspects, the sequence encoding the alphavirus target antigen comprises a sequence encoding a plurality of alphavirus target antigens. In some aspects, the sequence encoding a plurality of alphavirus target antigens comprises a plurality of gene inserts each corresponding to a target antigen and wherein each gene insert is separated by a nucleic acid sequence encoding a self-cleaving 2A peptide. In some aspects, the self-cleaving 2A peptide is derived from Porcine teschovirus-1 or Thosea asigna virus.

In some aspects, the replication defective virus vector is an adenovirus vector. In further aspects, the replication defective virus vector is an adenovirus 5 (Ad5) vector. In some aspects, the replication defective virus vector comprises an adenovirus vector with a deletion in an E1 gene region, an E2b gene region, an E3 gene region, E4 gene region, or any combination thereof. In further aspects, the deletion in the E2b gene region comprises a plurality of deletions in the E2b region. In some aspects, the deletion in the E1 gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof each comprises at least one base pair.

In other aspects, the deletion in the E1 gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof results from a translocation of two or more base pairs. In some aspects, the deletion in the E1 gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof each comprises at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, or at least 150 base pairs.

In other aspects, the deletion in the E1 gene region, the E2b gene region, the E3 gene region, the E4 gene region, or any combination thereof each comprises more than 150, more than 160, more than 170, more than 180, more than 190, more than 200, more than 250, or more than 300 base pairs.

In some aspects, the alphavirus target antigen comprises an antigen of a virus selected from the group consisting of Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), and Eastern equine encephalitis virus (EEEV), or any combination thereof. In some aspects, the alphavirus target antigen comprises an antigen of a virus selected from the group consisting of CHIKV, ONNV, RRV, and MAYV, or any combination thereof. In further aspects, the alphavirus target antigen comprises an antigen of CHIKV.

In some aspects, the alphavirus target antigen comprises an antigen selected from the group consisting of C, E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, and nsP4, or any combination thereof. In some aspects, the alphavirus target antigen comprises an antigen selected from the group consisting of C, E3_(ALPHA), E2_(ALPHA), and 6K, E1_(ALPHA), or any combination thereof. In some aspects, the alphavirus target antigen comprises an antigen selected from the group consisting of E1_(ALPHA) and E2_(ALPHA), or any combination thereof.

In some aspects, the sequence encoding an alphavirus target antigen comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, or any combination thereof.

In other aspects, the sequence encoding an alphavirus target antigen is an amino acid sequence, and wherein the amino acid sequence comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 21 or any combination thereof.

In some aspects, the sequence encoding an alphavirus target antigen is a nucleotide sequence, and wherein the nucleotide sequence comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 19, or any combination thereof. In some aspects, the sequence encoding an alphavirus target antigen comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or any combination thereof.

In some aspects, the replication defective virus vector further comprises an element to increase an expression of the alphavirus target antigen. In some aspects, the element comprises at least one element, at least 2 elements, at least 3 elements, at least 4 elements, or at least 5 elements.

In other aspects, the element comprises an internal ribosome binding site. In still other aspects, the element comprises a constitutive promoter. In some aspects, the element comprises an inducible promoter. In some aspects, the element comprises a transcription enhancer. In further aspects, the transcription enhancer is a Rous sarcoma virus (RSV) enhancer. In some aspects, the element does not contain a palindromic sequence.

In some aspects, the replication defective virus vector further comprises a nucleic acid sequence encoding a protein that increases alphavirus target antigen immunogenicity. In some aspects, the replication defective virus vector is not a gutted vector. In some aspects, the composition or the replication defective virus vector further comprises a sequence encoding a costimulatory molecule or an immunological fusion partner. In some aspects, the costimulatory molecule comprises B7, ICAM-1, LFA-3, or any combination thereof.

In various aspects, the present disclosure provides a pharmaceutical composition comprising the composition of any one of claims 1-34 and a pharmaceutically acceptable carrier.

In various aspects, the present disclosure provides a cell comprising any one of the above compositions. In some aspects, the cell is a host cell. In further aspects, the cell is a dendritic cell (DC).

In various aspects, the present disclosure provides a method of preparing a vaccine, comprising preparing any one of the above compositions or the above described pharmaceutical composition.

In various aspects, the present disclosure provides a method of generating an immune response against an alphavirus target antigen in a subject, comprising: administering to the subject any one of the above compositions or the above described pharmaceutical composition. In some aspects, the subject has not been infected with an alphavirus.

In some aspects, the alphavirus target antigen is from an alphavirus, wherein the alphavirus comprises Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), Eastern equine encephalitis virus (EEEV), or any combination thereof.

In various aspects, the present disclosure provides a method of preventing a Chikungunya virus infection in a subject, the method comprising administering to the subject a composition comprising: a replication defective virus vector comprising a deletion in an E2b gene region; and a nucleic acid sequence encoding at least one Chikungunya target antigen.

In some aspects, the subject has preexisting immunity to an adenovirus or an adenovirus vector. In some aspects, the subject is a human or a non-human animal. In some aspects, the administering is intravenously, subcutaneously, intralymphatically, intratumorally, intradermally, intramuscularly, intraperitoneally, intrarectally, intravaginally, intranasally, orally, via bladder instillation, or via scarification.

In some aspects, the administering of the composition to the subject is at least one time, is repeated at least twice, or is repeated at least three times. In some aspects, the administering to the subject comprises 1×10⁹ to 5×10¹² virus particles per dose. In some aspects, the administering to the subject comprises at least 10⁹ virus particles, at least 10¹⁰ virus particles, or at least 10¹¹ virus particles per dose. In some aspects, the replication defective virus vector is an adenovirus vector. In further aspects, the replication defective virus vector is an adenovirus 5 (Ad5) vector.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 exemplifies one structure of an alphavirus genome.

FIG. 2 exemplifies Ad5 [E1-, E2b-]-CHIKV_(str) vaccine derived from nucleotide positions 7567 to 11313 of SEQ ID NO: 1 that has been generated.

FIG. 3 exemplifies CHIKV structural protein expression in HEK 293 infected with Ad5 [E1-, E2b-]-CHIKV_(str) vaccine, or mock infected. CHIKV protein expression was detected by western blot.

FIG. 4 exemplifies results of a challenge study performed using Ad5 [E1-, E2b-]-CHIKV_(str) vaccine. Mice were vaccinated 2 times at days −14 and −7 and then challenged on day 0 with a lethal amount of CHIKV injected into the footpad.

FIG. 4A exemplifies mice vaccinated with Ad5 [E1-, E2b-]-CHIKV_(str) vaccine survive CHIKV infection.

FIG. 4B exemplifies mice vaccinated with Ad5 [E1-, E2b-]-CHIKV_(tr) vaccine maintain their pre-challenge weight.

FIG. 4C exemplifies mice vaccinated with Ad5 [E1-, E2b-]-CHIKV_(str) vaccine resolved CHIKV-induced footpad inflammation faster than the unvaccinated control mice.

FIG. 5 illustrates exemplary gene constructs for Chikungunya vaccines of the present disclosure.

FIG. 5A illustrates a schematic representation of a multiple gene construct containing three Chikungunya genes (C, E3, E2, 6K, and E1 gene (nucleotides 767-11313 of SEQ ID NO: 1); NS gene 1; NS gene 2) to be used for insertion into Ad5 [E1-, E2b-].

FIG. 5B illustrates the translation products of the gene construct in FIG. 5A and the stoichiometric abundance of each product.

FIG. 6 illustrates cell-mediated immune (CMI) responses (IFN-γ) and cytolytic T lymphocyte (CTL) responses and Granzyme B responses in splenocytes from immunized or control mice using an ELISpot assay. C57BL/6 mice were immunized two times at two-week intervals with 1×10¹⁰ virus particles (VPs) Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-null (empty vector control). One week after the final immunization, splenocytes from individual mice were tested for induction of immune responses after exposure of cells to Chikungunya virus peptides. The data show the cumulative number of spot forming cells (SFCs) per 10⁶ splenocytes after exposure to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays—CHIKV peptide pool 1 comprised peptides 1-103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310). Additional splenocytes were separately exposed to an SIV-nef peptide pools as a negative control prior to assay measurements.

FIG. 6A illustrates CMI responses in Ad5 [E1-, E2b-]-CHIK immunized and control C57BL/6 mice as measured by IFN-γ secreting SFCs using an ELISpot assay.

FIG. 6B illustrates CTL responses in Ad5 [E1-, E2b-]-CHIK immunized and control C57BL/6 mice as measured by Granzyme B (GR-B) secreting SFCs using an ELISpot assay.

FIG. 7 illustrates lymphocyte activation in splenocytes from immunized or control C57BL/6 mice as measured by intracellular expression of IFN-γ or IFN-γ/TNF-α analyzed by flow cytometry. C57BL/6 mice were immunized two times at two-week intervals with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-null (empty vector controls). One week after the final immunization, splenocytes from individual mice were exposed to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays—CHIKV peptide pool 1 comprised peptides 1-103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310) and analyzed by flow cytometry for induction of intracellular cytokine expression.

FIG. 7A illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).

FIG. 7B illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).

FIG. 7C illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ and TNF-α in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).

FIG. 7D illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ and TNF-α in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools).

FIG. 8 illustrates anti-Chikungunya envelope protein-2 antibody responses in immunized mice as compared to control mice as measured by a quantitative enzyme-linked immunosorbent assay (ELISA). C57BL/6 mice were immunized two times at two-week intervals with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-null (empty vector control). One week after the final immunization, sera from mice were evaluated for induction of antibody responses. Blood was collected by cheek pouch laceration under anesthesia.

DETAILED DESCRIPTION

The following passages describe different aspects in greater detail. Each aspect can be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

In certain embodiments, alphavirus antigens such as capsid, E3_(ALPHA), E2_(ALPHA), 6K, and E1_(ALPHA) and nonstructural proteins such as, nsP1, nsP2, nsP3, and nsP4 can be used, for example, in a vaccine composition or a composition comprising an adenovirus vector.

For example, E2_(ALPHA) and E1_(ALPHA) antigens can be used. Clinical correlates of protection have not been established for alphavirus vaccines, however there are data supporting a correlation between neutralizing antibody titers and protection. (Smalley, L., et al. Vaccine. (2016): 34(26): 2976-2981; Garcia-Arriaza, J. et al. Journal of Virology (2014): 88(6):3527-47).

Non-structural alphavirus antigens can also be used in certain aspects. Studies have shown that the non-structural proteins involved in replication of the genome contain conserved regions that can provide a wider range of alphavirus protection when used in experimental vaccines, including those employing Ad5 vectors.

As used herein, unless otherwise indicated, the article “a” means one or more unless explicitly otherwise provided for.

As used herein, unless otherwise indicated, terms such as “contain,” “containing,” “include,” “including,” and the like mean “comprising.”

As used herein, unless otherwise indicated, the term “or” can be conjunctive or disjunctive.

As used herein, unless otherwise indicated, any embodiment can be combined with any other embodiment.

As used herein, unless otherwise indicated, some inventive embodiments herein contemplate numerical ranges. A variety of aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range as if explicitly written out. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. When ranges are present, the ranges include the range endpoints.

The term “adenovirus” or “Ad” can refer to a group of non-enveloped DNA viruses from the family Adenoviridae. In addition to human hosts, these viruses can be found in, but are not limited to, avian, bovine, porcine and canine species. The use of any adenovirus from any of the four genera of the family Adenoviridae (e.g., Aviadenovirus, Mastadenovirus, Atadenovirus and Siadenovirus) can be contemplated as the basis of an E2b deleted virus vector, or vector containing other deletions as described herein. In addition, several serotypes are found in each species. Ad also pertains to genetic derivatives of any of these viral serotypes, including but not limited to, genetic mutation, deletion or transposition of homologous or heterologous DNA sequences.

A “helper adenovirus” or “helper virus” can refer to an Ad that can supply viral functions that a particular host cell cannot (the host can provide Ad gene products such as E1 proteins). This virus can be used to supply, in trans, functions (e.g., proteins) that are lacking in a second virus, or helper dependent virus (e.g., a gutted or gutless virus, or a virus deleted for a particular region such as E2b or other region as described herein); the first replication-incompetent virus can be said to “help” the second, helper dependent virus thereby permitting the production of the second viral genome in a cell.

The term “Adenovirus5 null (AdSnull),” as used herein, can refer to a non-replicating Ad that does not contain any heterologous nucleic acid sequences for expression.

The term “First Generation adenovirus,” as used herein, can refer to an Ad that has the early region 1 (E1) deleted. In additional cases, the nonessential early region 3 (E3) can also be deleted.

The term “gutted” or “gutless,” as used herein, can refer to an adenovirus vector that has been deleted of all viral coding regions.

The term “transfection” as used herein can refer to the introduction of foreign nucleic acid into eukaryotic cells. Transfection can be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” can refer to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell. The term “stable transfectant” can refer to a cell which has stably integrated foreign DNA into the genomic DNA.

The term “reporter gene” can indicate a nucleotide sequence that encodes a reporter molecule (including an enzyme). A “reporter molecule” can be detectable in any of a variety of detection systems, including, but not limited to enzyme-based detection assays (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems.

In one embodiment, the E. coli (3-galactosidase gene (available from Pharmacia Biotech, Pistacataway, N.J.), green fluorescent protein (GFP) (commercially available from Clontech, Palo Alto, Calif.), the human placental alkaline phosphatase gene, the chloramphenicol acetyltransferase (CAT) gene or other reporter genes that are known to the art can be employed.

As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” can refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides can determine the order of amino acids along the polypeptide (protein) chain. The nucleic acid sequence thus can code for the amino acid sequence.

The term “heterologous nucleic acid sequence,” as used herein, can refer to a nucleotide sequence that is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous nucleic acid can include a nucleotide sequence that is naturally found in the cell into which it is introduced or the heterologous nucleic acid can contain some modification relative to the naturally occurring sequence.

The term “transgene” can refer to any gene coding region, either natural or heterologous nucleic acid sequences or fused homologous or heterologous nucleic acid sequences, introduced into the cells or genome of a test subject. In the current invention, transgenes are carried on any viral vector that is used to introduce the transgcncs to the cells of the subject.

The term “Second Generation Adenovirus,” as used herein, can refer to an Ad that has all or parts of the E1, E2, E3, and, in certain embodiments, E4 DNA gene sequences deleted (removed) from the virus.

The term “subject,” or “individual,” as used herein, can refer to any animal, e.g., a mammal or marsupial. Subjects include but are not limited to humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats and fowl of any kind.

In certain aspects, there can be provided compositions and methods for producing a vaccine that generates immune responses against various alphaviruses using an adenovirus vector that allows for vaccinations to generate broadly reactive immune responses against alphaviruses.

One aspect provides a method of generating an immune response against one or more alphavirus target antigens in an individual comprising administering to the individual an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding one or more alphavirus target antigens; and readministering the adenovirus vector at least once or more to the individual; thereby generating an immune response against the alphavirus target antigens.

Another aspect provides a method for generating an immune response against several alphavirus target antigens in an individual, wherein the individual has preexisting immunity to adenovirus, comprising: administering to the individual an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding multiple alphavirus target antigens; and readministering the adenovirus vector at least once or more to the individual; thereby generating an immune response against the alphavirus target antigens.

I. Target Antigens

In certain aspects, the target antigens are comprised of antigens derived from various alphavirus proteins. In this regard, the alphavirus proteins can be derived from any alphavirus, including but not limited to Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), and Eastern equine encephalitis virus (EEEV). In certain embodiments, the at least one alphavirus virus protein can be an alphavirus protein comprising E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, or nsP4, or any combination thereof. In certain embodiments, the at least one alphavirus virus protein can comprise structural proteins from the group comprising E3_(ALPHA), E2_(ALPHA), 6K, or E1_(ALPHA), or any combination thereof. In certain embodiments, the at least one alphavirus virus protein can comprise non-structural proteins from the group comprising nsP1, nsP2, nsP3, or nsP4, or any combination thereof. In certain embodiments, the at least one alphavirus virus protein can comprise structural proteins from the group comprising E3_(ALPHA), E2_(ALPHA), 6K, or E1_(ALPHA), non-structural proteins from the group comprising nsP1, nsP2, nsP3, or nsP4, or combinations thereof. In certain embodiments, the at least one alphavirus virus protein can be an alphavirus protein selected from the group consisting of E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, and nsP4. In certain embodiments, the at least one alphavirus virus protein can comprise structural proteins selected from the group consisting of E3_(ALPHA), E2_(ALPHA), 6K, and E1_(ALPHA). In certain embodiments, the at least one alphavirus virus protein can comprise non-structural proteins selected from the group consisting of nsP1, nsP2, nsP3, and nsP4. In certain embodiments, the at least one alphavirus virus protein can comprise structural proteins selected from the group consisting of E3_(ALPHA), E2_(ALPHA), 6K, or E1_(ALPHA), non-structural proteins selected from the group consisting of nsP1, nsP2, nsP3, and nsP4, or combinations thereof.

The envelope glycoproteins E1_(ALPHA) and E2_(ALPHA) can form heterodimers that associate to form trimeric spikes on surface of the virion. The alphavirus replicative cycle can begin when the trimeric spikes bind host-cell receptors and can cause the endocytosis of virions. The low pH of the endosomes can induce fusion of the viral and endosomal membranes thereby releasing the viral genome into the cytosol of the cell.

The genomic RNA of alphaviruses can serve as an mRNA which, like cellular mRNAs, can be capped with 7-methylguanosine and can be polyadenylated. The first approximately 7 kB of the genome can encode the non-structural proteins that comprise the viral replicase and transcriptase. The final approximately 5 kB of the genome can encode the structural proteins. The viral replicase proteins, nsP1, nsP2, nsP3, and nsP4, produce anti-genome which then can serve as a template for production of genome and two mRNAs, one for the non-structural proteins and one for the structural proteins.

The non-structural proteins can be translated as a polyprotein that can be subsequently processed by nsP2. It is believed that differential processing of the polyprotein can be necessary for the switch between anti-genome and genome synthesis/sub-genomic mRNA synthesis.

The structural proteins can be translated as a polyprotein that can be processed by a combination of a serine protease activity on the capsid protein and cellular enzymes in the secretory system (e.g., signal peptidase and furin). The envelope glycoproteins, E1_(ALPHA) and E2_(ALPHA), can transit from the secretory system to the plasma membrane where they can be found in the fully processed, mature form. E1_(ALPHA) and E2_(ALPHA) recruit nucleocapsids (capsid protein shells with genomic RNA inside), and virions can be formed by budding from the plasma membrane.

Target antigens can include proteins, or variants or fragments thereof, associated with alphaviruses, such as C, E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, and nsP4. In some embodiments, the at least one target antigen is structural and/or non-structural antigen of an alphavirus. In certain embodiments, the at least one target antigen is any fragment of a protein or a polyprotein of an alphavirus. For example, the at least one target antigen used herein is a CHIKV structural antigen having an amino acid sequence set forth in SEQ ID NO: 2, a CHIKV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 3, or a combination thereof. In some embodiments, the at least one target antigen is a CHIKV antigen encoded by the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid sequence in the composition described herein comprises the CHIKV gene (nucleotides 7567-11313 of SEQ ID NO: 1) with a gene ID 956308, which has a gene symbol CHIKVgp2 and encodes a polyprotein containing C, E3, E2, 6K, and E1 proteins. In some embodiments, the nucleic acid sequence in the composition described herein comprises the CHIKV gene (nucleotides 77-7501 of SEQ ID NO: 1) with a gene ID 953609 which has a gene symbol CHIKVgp1 (encoding a polyprotein containing nsp1, nsp2, nsp3, and nsp4 proteins). In some embodiments, the nucleic acid sequence in the composition described herein comprises a nucleic acid sequence encoding a Chikungunya virus structural polyprotein (e.g., NP_690589) or fragments thereof. In some embodiments, the nucleic acid sequence in the composition described herein comprises a nucleic acid sequence encoding a Chikungunya virus nonstructural polyprotein (e.g., NP_690588) or fragments thereof. In some embodiments, the at least one target antigen is a CHIKV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the at least one target antigen is a CHIKV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, or any combination thereof.

For example, in some embodiments, the at least one target antigen is an ONNV antigen encoded by the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the at least one target antigen used herein is an ONNV structural antigen having an amino acid sequence set forth in SEQ ID NO: 5, an ONNV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 6, or a combination thereof. In some embodiments, the at least one target antigen is an ONNV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the at least one target antigen is a ONNV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, or any combination thereof. In some embodiments, the at least one target antigen is a MAYV antigen that is encoded by a sequence set forth in SEQ ID NO: 7. In other embodiments, the at least one target antigen used herein is a MAYV structural antigen having an amino acid sequence set forth in SEQ ID NO: 8, a MAYV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 9, or a combination thereof. In some embodiments, the at least one target antigen is a MAYV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the at least one target antigen is a MAYV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, or any combination thereof. In other embodiments, the at least one target antigen is a RRV antigen that is encoded by a sequence set forth in SEQ ID NO: 10. In some embodiments, the at least one target antigen used herein is a RRV structural antigen having an amino acid sequence set forth in SEQ ID NO: 11, a RRV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 12, or a combination thereof. In some embodiments, the at least one target antigen is a RRV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the at least one target antigen is a RRV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, or any combination thereof. In other embodiments, the at least one target antigen is a VEEV antigen that is encoded by a sequence set forth in SEQ ID NO: 13. In other embodiments, the at least one target antigen used herein is a VEEV structural antigen having an amino acid sequence set forth in SEQ ID NO: 14, a VEEV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 15, or a combination thereof. In some embodiments, the at least one target antigen is a VEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, the at least one target antigen is a VEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15, or any combination thereof. In other embodiments, the at least one target antigen is an EEEV antigen that is encoded by a sequence set forth in SEQ ID NO: 16. In some embodiments, the at least one target antigen used herein is an EEEV structural antigen having an amino acid sequence set forth in SEQ ID NO: 17, an EEEV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 18, or a combination thereof. In some embodiments, the at least one target antigen is a EEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, the at least one target antigen is a EEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, or any combination thereof. In some embodiments, the at least one target antigen is a WEEV antigen that is encoded by a sequence set forth in SEQ ID NO: 19. In other embodiments, the at least one target antigen used herein is a WEEV structural antigen having an amino acid sequence set forth in SEQ ID NO: 20, a WEEV non-structural antigen having an amino acid sequence set forth in SEQ ID NO: 21, or a combination thereof. In some embodiments, the at least one target antigen is a WEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 19. In some embodiments, the at least one target antigen is a WEEV antigen that is encoded by a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21, or any combination thereof.

II. Adenovirus Vectors

In certain aspects, adenovirus vectors can be used in compositions and methods for the delivery of alphavirus antigens.

The recombinant Ad5 [E1-, E2b-] vector vaccine platform can have additional deletions in the early gene 2b (E2b) region that remove the viral DNA polymerase (pol) and/or the pre-terminal protein (pTP) genes, and can be propagated in the E.C7 human cell line (Amalfitano A, Begy C R, Chamberlain J S Proc Natl Acad Sci USA. 1996 93:3352-6; Amalfitano A, Chamberlain J S Gene Ther. 1997 4:258-63; Amalfitano A et al. J Virol. 1998 72:926-33; Seregin S S and Amalfitano A Expert Opin Biol Ther. 2009 9:1521-31). The vector can be an expanded gene-carrying/cloning capacity of up to 12 kb, compared to the 7 kb capacity of current Ad5 [E1-] vectors, which can be sufficient to allow inclusion of multiple genes (Amalfitano A et al. J Virol. 1998 72:926-33; Seregin S S and Amalfitano A Expert Opin Biol Ther. 2009 9:1521-31). Additional deletions of the E2b region can confer advantageous immune properties such as eliciting potent immune responses to specific antigens while minimizing immune responses to Ad5 viral proteins.

Pre-clinical studies in animals and clinical studies in cancer demonstrate that Ad5 [E1-, E2b-]-based vectors can induce potent CMI and antibody (Ab) responses against a vectored antigen, even in the presence of Ad5 immunity.

The advanced recombinant adenovirus serotype 5 (Ad5) vector platforms can give the opportunity to develop a novel broadly cross-reactive vaccine for alphavirus. This vector can be delivered directly by subcutaneous injection for exposure of defined alphavirus antigens to antigen-presenting cells (APCs) that induce potent immune responses. Importantly, the Ad5 recombinant vector can replicate episomally and may not insert the genome into the host cell genome, thereby ensuring that there is no gene integration or disruption of vital cellular gene functions (Imler J L Vaccine. 1995 13:1143-51; Ertl H C, Xiang Z J Immunol. 1996 156:3579-82; Amalfitano, A Curr Opin Mol Ther. 2003 5:362-6).

Unfortunately, a major challenge facing current Ad5-based vectors is the presence of pre-existing immunity to Ad5. Most people exhibit neutralizing Abs against Ad5, the most widely used subtype for human vaccines, with two-thirds of people studied having lympho-proliferative responses against Ad5 (Chirmule N et al. Gene Ther. 1999 6:1574-83). This immunity can prevent the use of current early gene 1 (E1) region-deleted Ad5 vectors (Ad5 [E1-]) as a platform for an alphavirus vaccine. Ad5 immunity inhibits immunization, and especially re-immunization with recombinant Ad5 vectors, and can preclude immunization of a 15ittere against a second disease antigen as well. Overcoming the problem of pre-existing Ad5 vector immunity has been the subject of intense investigation. However, use of other Ad serotypes or even non-human forms of Ad can lead directly to altered production of important chemokines and cytokines, gene dysregulation, and can have significantly different biodistribution and tissue toxicities (Appledorn D M et al. Gene Ther. 2008 15:885-901; Hartman Z C et al. Virus Res. 2008 132:1-14). Even if these approaches succeed in an initial immunization, subsequent vaccinations can be problematic due to induced immune responses to the Ad subtype. To help avoid the Ad immunization barrier and circumvent the adverse conditions for current Ad5 [E1-] vectors, an improved Ad5 vector platform was constructed, described above.

Further, the Ad5 [E1-, E2b-] vectors can display reduced inflammation during the first 24 to 72 hours after injection compared to current Ad5 [E1-] vectors (Nazir S A, Metcalf J P J Investig Med. 2005 53:292-304; Schaack J Proc Natl Acad Sci USA. 2004 101:3124-9; Schaack J Viral Immunol. 2005 18:79-88). The lack of Ad5 [E1-, E2b-] late gene expression can render infected cells less vulnerable to anti-Ad5 activity and can permit them to produce and express the transgene for extended periods of time (Gabitzsch E S, Jones F R J Clin Cell Immunol. 2011 S4:001. Doi:10.4172/2155-9899. S4-001; Hodges B L J Gene Med. 2000 2:250-9). Reduced inflammatory responses against Ad5 [E1-, E2b-] viral proteins and the resulting evasion of pre-existing Ad5 immunity can increase the ability of Ad5 [E1-, E2b-] to infect APC cells, resulting in greater immunization of the inoculee. In addition, increased infection of other cell types can provide the high levels of antigen presentation needed for potent CD4⁺ and CD8⁺ T cell responses, leading to memory T cell development. Thus it appears that deletion of the E2b region can confer advantageous immune properties, such as eliciting potent immune responses to specific antigens, while minimizing immune responses to Ad5 proteins even in the presence of pre-existing Ad5 immunity.

Results demonstrated the ability of recombinant Ad5 [E1-, E2b-] platform-based vaccines to overcome pre-existing and/or Ad5 vector-induced immunity and induce significant protective immune responses. These studies established that new Ad5 [E1-, E2b-] vector-based vaccines 1) can induce significantly higher CMI responses compared to current Ad5 [E1-] vectors, 2) can be utilized for multiple immunization regimens designed to induce potent CMI responses, 3) can induce significant antigen-specific CMI responses in animals with pre-existing Ad5 immunity, and 4) can induce significant anti-tumor responses or protect against infectious disease in animals with high levels of pre-existing Ad5 immunity.

Certain aspects relate to methods and adenovirus vectors for generating immune responses against alphavirus target antigens. In particular, certain aspects can provide an improved Ad-based vaccine such that multiple vaccinations against more than one antigenic target entity can be achieved. Importantly, vaccination can be performed in the presence of preexisting immunity to the Ad and/or administered to subjects previously immunized multiple times with the adenovirus vector as described herein or other adenovirus vectors. The adenovirus vector can be administered to subjects multiple times to induce an immune response against a variety of alphavirus antigens, including but not limited to, the production of broad based antibody and cell-mediated immune responses against alphaviruses that cause polyarthralgias or encephalitis.

Certain aspects provide the use of E2b deleted adenovirus vectors, such as those described in U.S. Pat. Nos. 6,063,622; 6,451,596; 6,057,158: and 6,083,750 (all incorporated herein in their entirety by reference). As described in the '622 patent, in order to further cripple viral protein expression, and also to decrease the frequency of generating replication competent Ad (RCA), adenovirus vectors containing deletions in the E2b region can be provided in certain aspects. Propagation of these E2b deleted adenovirus vectors requires cell lines that can express the deleted E2b gene products.

In further aspects, there can be provided packaging cell lines; for example E.C7 (formally called C-7), derived from the HEK-203 cell line (Amalfitano A et al. Proc Natl Acad Sci USA 1996 93:3352-56; Amalfitano A et al. Gene Ther 1997 4:258-63).

Further, the E2b gene products, DNA polymerase and preterminal protein, can be constitutively expressed in E.C7, or similar cells along with the E1 gene products. Transfer of gene segments from the Ad genome to the production cell line can have immediate benefits: (1) increased carrying capacity of the recombinant DNA polymerase and preterminal protein-deleted adenovirus vector, since the combined coding sequences of the DNA polymerase and preterminal proteins that can be theoretically deleted approaches 4.6 kb; and, (2) a decreased potential of RCA generation, since two or more independent recombination events would be required to generate RCA.

Therefore, the E1, Ad DNA polymerase and preterminal protein expressing cell lines can enable the propagation of adenovirus vectors with a carrying capacity approaching 13 kb, without the need for a contaminating helper virus (Mitani et al. Proc. Natl. Acad. Sci. USA 1995 92:3854; Hodges et al. J Gene Med 2000 2:250-259; Amalfitano and Parks Curr Gene Ther 2002 2:111-133).

In addition, when genes critical to the viral life cycle are deleted (e.g., the E2b genes), a further crippling of Ad to replicate or express other viral gene proteins can occur. This can decrease immune recognition of virally infected cells, and can allow for extended durations of foreign transgene expression.

Important attributes of E1, DNA polymerase, and preterminal protein deleted vectors, however, include their inability to express the respective proteins from the E1 and E2b regions, as well as a predicted lack of expression of most of the viral structural proteins. For example, the major late promoter (MLP) of Ad is responsible for transcription of the late structural proteins L1 through L5 (Doerfler, In Adenovirus DNA, The Viral Genome and Its Expression (Martinus Nijhoff Publishing Boston, 1986)). Though the MLP is minimally active prior to Ad genome replication, the highly toxic Ad late genes are primarily transcribed and translated from the MLP only after viral genome replication has occurred (Thomas and Mathews Cell 1980 22:523). This cis-dependent activation of late gene transcription is a feature of DNA viruses in general, such as in the growth of polyoma and SV-40. The DNA polymerase and preterminal proteins are absolutely required for Ad replication (unlike the E4 or protein IX proteins) and thus their deletion is extremely detrimental to adenovirus vector late gene expression, and the toxic effects of that expression in cells such as APCs.

In certain embodiments, the adenovirus vectors contemplated for use include E2b deleted adenovirus vectors that have a deletion in the E2b region of the Ad genome and the E1 region but do not have any other regions of the Ad genome deleted. In another embodiment, the adenovirus vectors contemplated for use can include E2b deleted adenovirus vectors that have a deletion in the E2b region of the Ad genome and deletions in the E1 and E3 regions, but no other regions deleted. In a further embodiment, the adenovirus vectors contemplated for use can include adenovirus vectors that have a deletion in the E2b region of the Ad genome and deletions in the E1, E3 and partial or complete removal of the E4 regions but no other deletions.

In another embodiment, the adenovirus vectors contemplated for use include adenovirus vectors that have a deletion in the E2b region of the Ad genome and deletions in the E1 and E4 regions but no other deletions. In an additional embodiment, the adenovirus vectors contemplated for use can include adenovirus vectors that have a deletion in the E2a, E2b, and E4 regions of the Ad genome but no other deletions.

In one embodiment, the adenovirus vectors for use herein comprise vectors having the E1 and DNA polymerase functions of the E2b region deleted but no other deletions. In a further embodiment, the adenovirus vectors for use herein have the E1 and the preterminal protein functions of the E2b region deleted and no other deletions.

In another embodiment, the adenovirus vectors for use herein have the E1, DNA polymerase and the preterminal protein functions deleted, and no other deletions. In one particular embodiment, the adenovirus vectors contemplated for use herein are deleted for at least a portion of the E2b region and the E1 region, but are not “gutted” adenovirus vectors. In this regard, the vectors can be deleted for both the DNA polymerase and the preterminal protein functions of the E2b region.

The term “E2b deleted,” as used herein, can refer to a specific DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one E2b gene product. Thus, in certain embodiments, “E2b deleted” can refer to a specific DNA sequence that is deleted (removed) from the Ad genome. E2b deleted or “containing a deletion within the E2b region” can refer to a deletion of at least one base pair within the E2b region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In another embodiment, the deletion is of more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within the E2b region of the Ad genome. An E2b deletion can be a deletion that prevents expression and/or function of at least one E2b gene product and therefore, can encompass deletions within exons encoding portions of E2b-specific proteins as well as deletions within promoter and leader sequences. In certain embodiments, an E2b deletion is a deletion that prevents expression and/or function of one or both of the DNA polymerase and the preterminal protein of the E2b region. In a further embodiment, “E2b deleted” can refer to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations can include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein.

As would be understood by the skilled artisan upon reading the present disclosure, other regions of the Ad genome can be deleted. Thus to be “deleted” in a particular region of the Ad genome, as used herein, can refer to a specific DNA sequence that is mutated in such a way so as to prevent expression and/or function of at least one gene product encoded by that region. In certain embodiments, to be “deleted” in a particular region can refer to a specific DNA sequence that is deleted (removed) from the Ad genome in such a way so as to prevent the expression and/or the function encoded by that region (e.g., E2b functions of DNA polymerase or preterminal protein function). “Deleted” or “containing a deletion” within a particular region can refer to a deletion of at least one base pair within that region of the Ad genome. Thus, in certain embodiments, more than one base pair is deleted and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted from a particular region. In another embodiment, the deletion is more than 150, 160, 170, 180, 190, 200, 250, or 300 base pairs within a particular region of the Ad genome. In some embodiments, any one of the above described deletions can also be a result of translocation of two or more base pairs.

These deletions are such that expression and/or function of the gene product encoded by the region can be prevented. Thus deletions can encompass deletions within exons encoding portions of proteins as well as deletions within promoter and leader sequences. In a further embodiment, “deleted” in a particular region of the Ad genome can refer to one or more point mutations in the DNA sequence of this region of an Ad genome such that one or more encoded proteins is non-functional. Such mutations can include residues that are replaced with a different residue leading to a change in the amino acid sequence that result in a nonfunctional protein.

The adenovirus vectors comprising one or more deletions can be generated using recombinant techniques known in the art (see e.g., Amalfitano et al. J. Virol. 1998 72:926-33; Hodges, et al., J Gene Med 2000 2:250-59). As would be recognized by the skilled artisan, the adenovirus vectors for use can be successfully grown to high titers using an appropriate packaging cell line that constitutively expresses E2b gene products and products of any of the necessary genes that can have been deleted. In certain embodiments, HEK-293-derived cells that not only constitutively express the E1 and DNA polymerase proteins, but also the Ad-preterminal protein, can be used. In one embodiment, E.C7 cells are used to successfully grow high titer stocks of the adenovirus vectors (see e.g., Amalfitano et al. J. Virol. 1998 72:926-33; Hodges et al. J Gene Med 2000 2:250-59)

In order to delete critical genes from self-propagating adenovirus vectors, the proteins encoded by the targeted genes have to first be coexpressed in HEK-293 cells, or similar, along with the E1 proteins. Therefore, only those proteins which are non-toxic when coexpressed constitutively (or toxic proteins inducibly-expressed) can be utilized. Coexpression in HEK-293 cells of the E1 and E4 genes has been demonstrated (utilizing inducible, not constitutive, promoters) (Yeh et al. J. Virol. 1996 70:559; Wang et al. Gene Therapy 1995 2:775; and Gorziglia et al. J. Virol. 1996 70:4173). The E1 and protein IX genes (a virion structural protein) have been coexpressed (Caravokyri and Leppard J. Virol. 1995 69:6627), and coexpression of the E1, E4, and protein IX genes has also been described (Krougliak and Graham Hum. Gene Ther. 1995 6:1575). The E1 and 100 k genes have been successfully expressed in transcomplementing cell lines, as have E1 and protease genes (Oualikene et al. Hum Gene Ther 2000 11:1341-53; Hodges et al. J. Virol 2001 75:5913-20).

Cell lines coexpressing E1 and E2b gene products for use in growing high titers of E2b deleted Ad particles are described in U.S. Pat. No. 6,063,622. The E2b region can encode the viral replication proteins which are absolutely required for Ad genome replication (Doerfler, supra and Pronk et al. Chromosoma 1992 102:S39-S45). Useful cell lines constitutively express the approximately 140 kDa Ad-DNA polymerase and/or the approximately 90 kDa preterminal protein. In particular, cell lines that have high-level, constitutive coexpression of the E1, DNA polymerase, and preterminal proteins, without toxicity (e.g., E.C7), can be desirable for use in propagating Ad for use in multiple vaccinations. These cell lines can permit the propagation of adenovirus vectors deleted for the E1, DNA polymerase, and preterminal proteins.

The recombinant Ad can be propagated using techniques known in the art. For example, in certain embodiments, tissue culture plates containing E.C7 cells are infected with the adenovirus vector virus stocks at an appropriate MOI (e.g., 5) and incubated at 37.0° C. for 40-96 h. The infected cells can be harvested, resuspended in 10 mM Tris-CI (pH 8.0), and sonicated, and the virus can be purified by two rounds of cesium chloride density centrifugation. In certain techniques, the virus containing band is desalted over a Sephadex CL-6B column (Pharmacia Biotech, Piscataway, N.J.), sucrose or glycerol is added, and aliquots are stored at −80° C. In some embodiments, the virus can be placed in a solution designed to enhance its stability, such as A195 (Evans et al. J Pharm Sci 2004 93:2458-75). The titer of the stock can be measured (e.g., by measurement of the optical density at 260 nm of an aliquot of the virus after SDS lysis). In another embodiment, plasmid DNA, either linear or circular, encompassing the entire recombinant E2b deleted adenovirus vector can be transfected into E.C7, or similar cells, and can be incubated at 37.0° C. until evidence of viral production is present (e.g., the cytopathic effect). The conditioned media from these cells can then be used to infect more E.C7, or similar cells, to expand the amount of virus produced, before purification.

Purification can be accomplished by two rounds of cesium chloride density centrifugation or selective filtration. In certain embodiments, the virus can be purified by column chromatography, using commercially available products (e.g., Adenopure from Puresyn, Inc., Malvem, Pa.) or custom made chromatographic columns.

In certain embodiments, the recombinant Ad can comprise enough of the virus to ensure that the cells to be infected are confronted with a certain number of viruses. Thus, there can be provided a stock of recombinant Ad, particularly, an RCA-free stock of recombinant Ad. The preparation and analysis of Ad stocks is well known in the art. Viral stocks can vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them. The viral stocks can have a titer of at least about 10⁶, 10⁷, or 10⁸ virus particles (VPs)/ml, and many such stocks can have higher titers, such as at least about 10⁹, 10¹⁰, 10¹¹, or 10¹² VPs/ml.

III. Heterologous Nucleic Acids

In certain embodiments, the adenovirus vectors described herein comprise heterologous nucleic acid sequences that encode one or more target antigens of interest such as alphavirus target antigens, fragments or fusions thereof, against which it is desired to generate an immune response. In some embodiments, the adenovirus vectors comprise heterologous nucleic acid sequences that encode several proteins, fusions thereof or fragments thereof, which can modulate the immune response. Certain aspects provide the Second Generation E2b deleted adenovirus vectors that comprise a heterologous nucleic acid sequence such as an alphavirus target antigen.

As such, certain aspects provide nucleic acid sequences, which can also be referred to herein as polynucleotides that encode several alphavirus target antigens of interest. As such, certain aspects provide polynucleotides that encode target antigens from any source as described further herein, vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors. The terms “nucleic acid” and “polynucleotide” are used essentially interchangeably herein. As will be also recognized by the skilled artisan, polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules can include hnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences can, but need not, be present within a polynucleotide, and a polynucleotide can, but need not, be linked to other molecules and/or support materials. An isolated polynucleotide, as used herein, can mean that a polynucleotide is substantially away from other coding sequences. For example, an isolated DNA molecule as used herein does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this can refer to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment recombinantly in the laboratory.

As will be understood by those skilled in the art, the polynucleotides can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or can be adapted to express target antigens as described herein, fragments of antigens, peptides and the like. Such segments can be naturally isolated, or modified synthetically by the hand of man.

Polynucleotides can comprise a native sequence (i.e., an endogenous sequence that encodes a target antigen polypeptide/protein/epitope or a portion thereof) or can comprise a sequence that encodes a variant or derivative of such a sequence. In certain embodiments, the polynucleotide sequences set forth herein encode target antigen proteins as described herein. In some embodiments, polynucleotides represent a novel gene sequence that has been optimized for expression in specific cell types (i.e., human cell lines) that can substantially vary from the native nucleotide sequence or variant but encode a similar protein antigen.

In other related embodiments, there can be provided polynucleotide variants having substantial identity to native sequences encoding proteins (e.g., target antigens of interest) as described herein, for example those comprising at least 70% sequence identity, particularly at least 75% up to 99% or higher, sequence identity compared to a native polynucleotide sequence encoding the polypeptides using the methods described herein (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

In certain aspects, polynucleotide variants can contain one or more substitutions, additions, deletions and/or insertions, particularly such that the immunogenicity of the epitope of the polypeptide encoded by the variant polynucleotide or such that the immunogenicity of the heterologous target protein may not be substantially diminished relative to a polypeptide encoded by the native polynucleotide sequence. As described elsewhere herein, the polynucleotide variants can encode a variant of the target antigen, or a fragment (e.g., an epitope) thereof wherein the propensity of the variant polypeptide or fragment (e.g., epitope) thereof to react with antigen-specific antisera and/or T-cell lines or clones may not be substantially diminished relative to the native polypeptide. The term “variants” can also encompass homologous genes of xenogeneic origin.

Certain aspects provide polynucleotides that comprise or consist of at least about 5 up to a 1000 or more contiguous nucleotides encoding a polypeptide, including target protein antigens, as described herein, as well as all intermediate lengths there between. It will be readily understood that “intermediate lengths,” in this context, can mean any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like. A polynucleotide sequence as described herein can be extended at one or both ends by additional nucleotides not found in the native sequence encoding a polypeptide as described herein, such as an epitope or heterologous target protein. This additional sequence can consist of 1 up 20 nucleotides or more, at either end of the disclosed sequence or at both ends of the disclosed sequence.

In certain embodiments, the polynucleotides, or fragments thereof, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, expression control sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length can vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length can be employed, and the total length that can be limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative polynucleotide segments with total lengths of about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations.

When comparing polynucleotide sequences, two sequences can be said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences can be performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison can be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff M O (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff M O (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990); Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins D G and Sharp P M CABIOS 1989 5:151-53; Myers E W and Muller W CABIOS 1988 4:11-17; Robinson E D Comb. Theor 1971 11A 05; Saitou N, Nei M MoI. Biol. Evol. 1987 4:406-25; Sneath P H A and Sokal R R Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif. (1973); Wilbur W J and Lipman D J Proc. Natl. Acad., Sci. USA 1983 80:726-30).

Alternatively, optimal alignment of sequences for comparison can be conducted by the local identity algorithm of Smith and Waterman, Add. APL. Math 1981 2:482, by the identity alignment algorithm of Needleman and Wunsch J. MoI. Biol. 1970 48:443, by the search for similarity methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

One example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nucl. Acids Res. 1977 25:3389-3402, and Altschul et al. J. MoI. Biol. 1990 215:403-10, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction can be halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, and expectation I of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 1989 89:10915) alignments, (B) of 50, expectation I of 10, M=5, N=−4 and a comparison of both strands.

In certain aspects, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there can be many nucleotide sequences that encode a particular antigen of interest, or fragment thereof, as described herein. Some of these polynucleotides can bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated in certain aspects. Further, alleles of the genes comprising the polynucleotide sequences provided herein are also contemplated. Alleles can be endogenous genes that can be altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein can, but need not, have an altered structure or function. Alleles can be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

Therefore, in another embodiment, a mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of variants and/or derivatives of the target antigen sequences, or fragments thereof, as described herein. By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques can provide a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

Site-specific mutagenesis can allow the production of mutants through the use of specific oligonucleotide sequences which can encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations can be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.

Polynucleotide segments or fragments encoding the polypeptides can be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments can be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Pat. No. 4,683,202, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology (see for example, Current Protocols in Molecular Biology, John Wiley and Sons, NY, NY).

In order to express a desired target antigen polypeptide or fragment thereof, or fusion protein comprising any of the above, as described herein, the nucleotide sequences encoding the polypeptide, or functional equivalents, can be inserted into an appropriate Ad as described elsewhere herein using recombinant techniques known in the art. The appropriate adenovirus vector can contain the necessary elements for the transcription and translation of the inserted coding sequence and any desired linkers. Methods that are well known to those skilled in the art can be used to construct these adenovirus vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods can include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Amalfitano et al. J. Virol. 1998 72:926-33; Hodges et al. J Gene Med 2000 2:250-259; Sambrook J et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel F M et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of vector/host systems can be utilized to contain and produce polynucleotide sequences. These can include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA vectors; yeast transformed with yeast vectors; insect cell systems infected with virus vectors (e.g., baculovirus); plant cell systems transformed with virus vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in an adenovirus vector can be those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which can interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, sequences encoding a polypeptide of interest can be ligated into an Ad transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing the polypeptide in infected host cells (Logan J and Shenk T (1984) Proc. Natl. Acad. Sci 1984 87:3655-59). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells. Transcriptional enhancers can comprise one element, at least two elements, at least three elements, at least four elements, at least five elements, or at least six elements.

Specific initiation signals can also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals can include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon can be provided. Furthermore, the initiation codon can be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers that are appropriate for the particular cell system which is used, such as those described in the literature (Scharf D. et al. Results Probl. Cell Differ. 1994 20:125-62). Specific termination sequences, either for transcription or translation, can also be incorporated in order to achieve efficient translation of the sequence encoding the polypeptide of choice.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products (e.g., target antigens of interest), using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide can be used for some applications, but a competitive binding assay can also be employed. These and other assays are described, among other places, in Hampton R et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox D E et al. J. Exp. Med. 1983 758:1211-16). The adenovirus vectors can comprise nucleic acid sequences encoding several alphavirus antigens of interest.

In certain embodiments, elements that increase the expression of the desired target antigen are incorporated into the nucleic acid sequence of the adenovirus vectors described herein. Such elements include internal ribosome binding sites (IRES; Wang and Siddiqui Curr. Top. Microbiol. Immunol 1995 203:99; Ehrenfeld and Semler Curr. Top. Microbiol. Immunol. 1995 203:65; Rees et al., Biotechniques 1996 20:102; Sugimoto et al. Biotechnology 1994 2:694). IRES can increase translation efficiency. Other sequences can also enhance expression. For some genes, sequences especially at the 5′ end can inhibit transcription and/or translation. These sequences can be palindromes that can form hairpin structures. Any such sequences in the nucleic acid to be delivered can be deleted or not deleted.

Expression levels of the transcript or translated product can be assayed to confirm or ascertain which sequences affect expression. Transcript levels can be assayed by any known method, including Northern blot hybridization, Rnase probe protection and the like. Protein levels can be assayed by any known method, including ELISA. As would be recognized by the skilled artisan, the adenovirus vectors comprising heterologous nucleic acid sequences can be generated using recombinant techniques known in the art, such as those described in Maione et al. Proc Natl Acad Sci USA 2001 98:5986-91; Maione et al. Hum Gene Ther 2000 1:859-68; Sandig et al. Proc Natl Acad Sci USA, 2000 97:1002-07; Harui et al. Gene Therapy 2004 11:1617-26; Parks et al. Proc Natl Acad Sci USA 1996 93:13565-570; Dello Russo et al. Proc Natl Acad Sci USA 2002 99:12979-984; Current Protocols in Molecular Biology, John Wiley and Sons, NY, NY).

As noted above, the adenovirus vectors can comprise nucleic acid sequences that can encode several alphavirus target proteins or antigens of interest. In this regard, the vectors can contain nucleic acid encoding 1 to 4 or more different target antigens of interest. The target antigens can be a full length protein or can be a fragment (e.g., an epitope) thereof. The adenovirus vectors can contain nucleic acid sequences encoding multiple fragments or epitopes from one target protein of interest or can contain one or more fragments or epitopes from numerous different target alphavirus antigen proteins of interest.

In some aspects, the nucleic acid sequences encode a plurality of alphavirus target antigens. The nucleic acid sequence encoding the plurality of alphavirus target antigens can comprise a plurality of gene inserts each corresponding to a target antigen and wherein each gene insert is separated by a nucleic acid sequence encoding a self-cleaving 2A peptide. In some aspects, the self-cleaving 2A peptide (i.e., the cleavable linker) is derived from Porcine teschovirus-1 or Thosea asigna virus or the like.

Examples of cleavable linkers can include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picornaviral 2A-like linker, CHYSEL sequences of Porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof.

In certain embodiments, immunogenic fragments bind to an MHC class I or class II molecule. As used herein, an immunogenic fragment can “bind to” an MHC class I or class II molecule if such binding is detectable using any assay known in the art. For example, the ability of a polypeptide to bind to MHC class I can be evaluated indirectly by monitoring the ability to promote incorporation of ¹²⁵I labeled 32-microglobulin (02m) into MHC class V/32m/peptide heterotrimeric complexes (see Parker et al., J. Immunol. 752:163, 1994). Alternatively, functional peptide competition assays that are known in the art can be employed. Immunogenic fragments of polypeptides can generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3^(rd) ed., 243-247 (Raven Press, 1993) and references cited therein. Representative techniques for identifying immunogenic fragments can include screening polypeptides for the ability to react with antigen-specific antisera and/or T-cell lines or clones. An immunogenic fragment of a particular target polypeptide can be a fragment that can react with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length target polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). In other words, an immunogenic fragment can react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens can generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Target antigens can include but are not limited to antigens derived from any of the alphaviruses. Target antigens can include proteins produced by any of the infectious alphaviruses described herein, such as, C, E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, and nsP4. As used herein, an “infectious agent” can be any species capable of infecting a host. Infectious agents can include, for example, any virus within the alphavirus genus.

The adenovirus vector can also include nucleic acid sequences that encode proteins that increase the immunogenicity of the target antigen. In this regard, the protein produced following immunization with the adenovirus vector containing such a protein can be a fusion protein comprising the target antigen of interest fused to a protein that increases the immunogenicity of the target antigen of interest.

IV. Combination Therapies

Certain embodiments provide a combination immunotherapy and vaccine composition for the treatment and prevention infectious diseases. Some embodiments provide combination multi-targeted vaccines, immunotherapies, and methods for enhanced therapeutic response to complex diseases such as infectious diseases. Each component of the combination therapy can be independently included in a vaccine composition for prevention of Chikungunya infection or infection by any alphavirus.

“Treatment” can refer to administration of a therapeutically effective dose of a vaccine of this disclosure to a subject. The treatment can be administered in a pharmaceutical composition to a subject. The subject can also be healthy and disease free at the time of treatment and, in this case, the treatment can be referred to as a preventative vaccination. The subject can be suffering from a disease condition at the time of treatment and, in this case, the treatment can be referred to as therapeutic vaccination.

A “subject” can refer to any animal, including, but not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats and fowls. A “subject” can be used herein interchangeably with “individual” or “patient.”

In some aspects, the vector comprises at least one antigen. In some aspects, the vector comprises at least two antigens. In some aspects, the vaccine formulation comprises 1:1 ratio of vector to antigen. In some aspects, the vaccine comprises 1:2 ratio of vector to antigen. In some aspects, the vaccine comprises 1:3 ratio of vector to antigen. In some aspects, the vaccine comprises 1:4 ratio of vector to antigen. In some aspects, the vaccine comprises 1:5 ratio of vector to antigen. In some aspects, the vaccine comprises 1:6 ratio of vector to antigen. In some aspects, the vaccine comprises 1:7 ratio of vector to antigen. In some aspects, the vaccine comprises 1:8 ratio of vector to antigen. In some aspects, the vaccine comprises 1:9 ratio of vector to antigen. In some aspects, the vaccine comprises 1:10 ratio of vector to antigen.

In some aspects, the vaccine is a combination vaccine, wherein the vaccine comprises at least two vectors each containing at least a single antigen.

When a mixture of different antigens are simultaneously administered or expressed from a same or different vector in a subject, they can compete with one another. As a result the formulations comprising different concentration and ratios of expressed antigens in a combination immunotherapy or vaccine must be evaluated and tailored to the subject or group of subjects to ensure that effective and sustained immune responses occur after administration.

Composition that comprises multiple antigens can be present at various ratios. For example, formulations with more than one vector can have various ratios. For example, immunotherapies or vaccines can have two different vectors in a stoichiometry of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 2:1, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:1, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 4:1,4:3, 4:5, 4:6, 4:7, 4:8, 5:1, 5:3, 5:4, 5:6, 5:7, 5:8, 6:1, 6:3, 6:4, 6:5, 6:7, 6:8, 7:1, 7:3, 7:4, 7:5, 7:6, 7:8, 8:1, 8:3, 8:4, 8:5, 8:6, or 8:7.

In some embodiments, at least one of the recombinant nucleic acid vectors is a replication defective virus vector that comprises a replication defective adenovirus 5 vector comprising a first identity value. In some embodiments, the replication defective adenovirus vector comprises a deletion in the E2b gene region. In some embodiments, the replication defective adenovirus vector further comprises a deletion in the E1 gene region. In some embodiments, the replication defective adenovirus vector comprises a deletion in an E1 gene region, and E2b gene region, an E3 gene region, an E4 gene region, or any combination thereof.

Specific therapies that can be used in combination with any Ad5 [E1-, E2b-] vaccine of the present disclosure are described in further detail below.

A. Costimulatory Molecules

In addition to the use of a recombinant adenovirus-based vector vaccine containing target antigens such as alphavirus target antigens, particularly Chikungunya antigens, co-stimulatory molecules can be incorporated into said the vaccine to increase immunogenicity.

Initiation of an immune response can require at least two signals for the activation of I T cells by APCs (Damle, et al. J Immunol 148:1985-92 (1992); Guinan, et al. Blood 84:3261-82 (1994); Hellstrom, et al. Cancer Chemother Pharmacol 38:S40-44 (1996); Hodge, et al. Cancer Res 39:5800-07 (1999). An antigen specific first signal can be delivered through the T cell receptor (TCR) via the peptide/major histocompatibility complex (MHC) and can cause the T cell to enter the cell cycle. A second, or costimulatory, signal can be delivered for cytokine production and proliferation.

At least three distinct molecules normally found on the surface of professional antigen presenting cells (APCs) can be capable of providing the second signal critical for T cell activation: B7-1 (CD80), ICAM-1 (CD54), and LFA-3 (human CD58) (Damle, et al. J Immunol 148:1985-92 (1992); Guinan, et al. Blood 84: 3261-82 (1994); Wingren, et al. Crit Rev Immunol 15: 235-53 (1995); Parra, et al. Scand. J Immunol 38: 508-14 (1993); Hellstrom, et al. Ann NY Acad Sci 690: 225-30 (1993); Parra, et al. J Immunol 158: 637-42 (1997); Sperling, et al. J Immunol 157: 3909-17 (1996); Dubey, et al. J Immunol 155: 45-57 (1995); Cavallo, et al. Eur J Immunol 25: 1154-62 (1995).

These costimulatory molecules can have distinct T cell ligands. B7-1 can interact with the CD28 and CTLA-4 molecules, ICAM-1 can interact with the CD11a/CD18 (LFA-1/beta-2 integrin) complex, and LFA-3 can interact with the CD2 (LFA-2) molecules. Therefore, in a certain embodiment, it would be desirable to have a recombinant adenovirus vector that contains B7-1, ICAM-1, and LFA-3, respectively, that, when combined with a recombinant adenovirus-based vector vaccine containing one or more nucleic acids encoding target antigens such as alphavirus antigens, can further increase/enhance anti-alphavirus immune responses directed to specific target antigens.

V. Immunological Fusion Partners

The viral vector or composition described herein can further comprise nucleic acid sequences that encode proteins, or an “immunological fusion partner,” that can increase the immunogenicity of the target antigen such as Chikungunya virus antigens, or any target antigen of the present disclosure. In this regard, the protein produced following immunization with the viral vector containing such a protein can be a fusion protein comprising the target antigen of interest fused to a protein that increases the immunogenicity of the target antigen of interest. Furthermore, combination therapy with Ad5[E1-, E2b-] vectors encoding for a Chikungunya virus antigen and an immunological fusion partner can result in boosting the immune response, such that the combination of both therapeutic moieties acts to synergistically boost the immune response more than either the Ad5[E1-, E2b-] vector encoding for a Chikungunya virus antigen alone, or the immunological fusion partner alone. For example, combination therapy with an Ad5[E1-, E2b-] vector encoding for a Chikungunya virus antigen and an immunological fusion partner can result in synergistic enhancement of stimulation of antigen-specific effector CD4+ and CD8+ T cells, stimulation of NK cell response directed towards killing infected cells, stimulation of neutrophils or monocyte cell responses directed towards killing infected cells via antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) mechanisms, or any combination thereof. This synergistic boost can vastly improve survival outcomes after administration to a subject in need thereof. In certain embodiments, combination therapy with an Ad5[E1-, E2b-] vector encoding for a Chikungunya virus antigen and an immunological fusion partner can result in generating an immune response comprises an increase in target antigen-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the adenovirus vectors as compared to a control. In another embodiment, generating an immune response comprises an increase in target-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the Ad5[E1-, E2b-] vector encoding for a Chikungunya virus antigen and an immunological fusion partner as compared to a control. In a further embodiment, generating an immune response that comprises an increase in target antigen-specific cell-mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamma (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), or other cytokines, of about 1.5 to 20, or more fold as compared to a control. In a further embodiment, generating an immune response comprises an increase in target-specific antibody production of between 1.5 and 5 fold in a subject administered the Ad5[E1-, E2b-] vectors encoding for a Chikungunya virus antigen and an immunological fusion partner as described herein as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target-specific antibody production of about 1.5 to 20, or more fold in a subject administered the adenovirus vector as compared to a control.

As an additional example, combination therapy with an Ad5[E1-, E2b-] vector encoding for target epitope antigens and an immunological fusion partner can result in synergistic enhancement of stimulation of antigen-specific effector CD4+ and CD8+ T cells, stimulation of NK cell response directed towards killing infected cells, stimulation of neutrophils or monocyte cell responses directed towards killing infected cells via antibody dependent cell-mediated cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) mechanisms, or any combination thereof. This synergistic boost can vastly improve survival outcomes after administration to a subject in need thereof. In certain embodiments, combination therapy with Ad5[E1-, E2b-] vectors encoding for a target epitope antigen and an immunological fusion partner can result in generating an immune response comprises an increase in target antigen-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the adenovirus vectors as compared to a control. In another embodiment, generating an immune response comprises an increase in target-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the Ad5[E1-, E2b-] vector encoding for target epitope antigen and an immunological fusion partner as compared to a control. In a further embodiment, generating an immune response that comprises an increase in target antigen-specific cell-mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamma (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), or other cytokines, of about 1.5 to 20, or more fold as compared to a control. In a further embodiment, generating an immune response comprises an increase in target-specific antibody production of between 1.5 and 5 fold in a subject administered the adenovirus vectors as described herein as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target-specific antibody production of about 1.5 to 20, or more fold in a subject administered the adenovirus vector as compared to a control.

In one embodiment, such an immunological fusion partner is derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ral2 fragment. The immunological fusion partner derived from Mycobacterium sp. can be any one of the sequences set forth in SEQ ID NO: 22-SEQ ID NO: 30 and SEQ ID NO: 93-SEQ ID NO: 98. Oligonucleotides, Met-His tags, and enterokinase recognition sites, which can be used to construct these Mycobacterium sp.-derived Ral2 sequences are set forth in any one of SEQ ID NO: 99-SEQ ID NO: 106 as shown in TABLE 2. Ral2 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences are described in U.S. Pat. No. 7,009,042, which is herein incorporated by reference in its entirety. Briefly, Ral2 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is a serine protease of 32 kDa encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (see, e.g., U.S. Pat. No. 7,009,042; Skeiky et al., Infection and Immun. 67:3998-4007 (1999), incorporated herein by reference in their entirety). C-terminal fragments of the MTB32A coding sequence can be expressed at high levels and remain as soluble polypeptides throughout the purification process. Moreover, Ral2 can enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. A Ral2 fusion polypeptide can comprise a 14 kDa C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other Ral2 polynucleotides generally can comprise at least about 15, 30, 60, 100, 200, 300, or more nucleotides that encode a portion of a Ral2 polypeptide. Ral2 polynucleotides can comprise a native sequence (i.e., an endogenous sequence that encodes a Ra12 polypeptide or a portion thereof) or can comprise a variant of such a sequence. Ral2 polynucleotide variants can contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ral2 polypeptide. Variants can have at least about 70%, 80%, or 90% identity, or more, to a polynucleotide sequence that encodes a native Ral2 polypeptide or a portion thereof.

In certain aspects, an immunological fusion partner can be derived from protein D, a surface protein of the gram-negative bacterium Haemophilus 34ittered34 B. The immunological fusion partner derived from protein D can be the sequence set forth in SEQ ID NO: 31. In some cases, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids). A protein D derivative can be 34ittered34. Within certain embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes, which can increase the expression level in E. coli and can function as an expression enhancer. The lipid tail can ensure optimal presentation of the antigen to antigen presenting cells. Other fusion partners can include the non-structural protein from influenza virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes can be used.

In certain aspects, the immunological fusion partner can be the protein known as LYTA, or a portion thereof (particularly a C-terminal portion). The immunological fusion partner derived from LYTA can the sequence set forth in SEQ ID NO: 32. LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein can be responsible for the affinity to the choline or to some choline analogues such as DEAE. This property can be exploited for the development of E. coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus can be employed. Within another embodiment, a repeat portion of LYTA can be incorporated into a fusion polypeptide. A repeat portion can, for example, be found in the C-terminal region starting at residue 178. One particular repeat portion can incorporate residues 188-305.

In some embodiments, the target antigen is fused to an immunological fusion partner, which can also be referred to herein as an “immunogenic component,” comprising a cytokine selected from the group of IFN-γ, TNFα, IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF-1), IFN-α, IFN-β, IL-1α, IL-1β, IL-1RA, IL-11, IL-17A, IL-17F, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31, IL-33, IL-34, IL-35, IL-36α,β,λ, IL-36Ra, IL-37, TSLP, LIF, OSM, LT-α, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, OPG-L, APRIL, LIGHT, TWEAK, BAFF, TGF-β1, and MIF. The target antigen fusion can produce a protein with substantial identity to one or more of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF-1), IFN-α, IFN-β, IL-1α, IL-1β, IL-1RA, IL-11, IL-17A, IL-17F, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31, IL-33, IL-34, IL-35, IL-36α,β,λ, IL-36Ra, IL-37, TSLP, LIF, OSM, LT-α, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, OPG-L, APRIL, LIGHT, TWEAK, BAFF, TGF-β1, and MIF. The target antigen fusion can encode a nucleic acid encoding a protein with substantial identity to one or more of IFN-γ, TNFα, IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF-1), IFN-α, IFN-3, IL-1α, IL-β, IL-1RA, IL-11, IL-17A, IL-17F, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31, IL-33, IL-34, IL-35, IL-36α,β,λ, IL-36Ra, IL-37, TSLP, LIF, OSM, LT-α, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, OPG-L, APRIL, LIGHT, TWEAK, BAFF, TGF-β1, and MIF. In some embodiments, the target antigen fusion further comprises one or more immunological fusion partner, which can also be referred to herein as an “immunogenic components,” comprising a cytokine selected from the group of IFN-γ, TNFα, IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF-1), IFN-α, IFN-β, IL-1α, IL-1β, IL-1RA, IL-11, IL-17A, IL-17F, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31, IL-33, IL-34, IL-35, IL-36α,β,λ, IL-36Ra, IL-37, TSLP, LIF, OSM, LT-α, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, OPG-L, APRIL, LIGHT, TWEAK, BAFF, TGF-β1, and MIF. The sequence of IFN-γ can be, but is not limited to, a sequence as set forth in SEQ ID NO: 33. The sequence of TNFα can be, but is not limited to, a sequence as set forth in SEQ ID NO: 34. The sequence of IL-2 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 35. The sequence of IL-8 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 36. The sequence of IL-12 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 37. The sequence of IL-18 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 38. The sequence of IL-7 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 39. The sequence of IL-3 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 40. The sequence of IL-4 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 41. The sequence of IL-5 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 42. The sequence of IL-6 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 43. The sequence of IL-9 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 44. The sequence of IL-10 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 45. The sequence of IL-13 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 46. The sequence of IL-15 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 47. The sequence of IL-16 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 74. The sequence of IL-17 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 75. The sequence of IL-23 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 76. The sequence of IL-32 can be, but is not limited to, a sequence as set forth in SEQ ID NO: 77.

In some embodiments, the target antigen is fused or linked to an immunological fusion partner, also referred to herein as an “immunogenic component,” comprising a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF-1), IFN-α, IFN-β3, IL-1α, IL-β, IL-1RA, IL-11, IL-17A, IL-17F, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31, IL-33, IL-34, IL-35, IL-36α,β,λ, IL-36Ra, IL-37, TSLP, LIF, OSM, LT-α, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, OPG-L, APRIL, LIGHT, TWEAK, BAFF, TGF-β1, and MIF. In some embodiments, the target antigen is co-expressed in a cell with an immunological fusion partner, also referred to herein as an “immunogenic component,” comprising a cytokine selected from the group of IFN-γ, TNFα IL-2, IL-8, IL-12, IL-18, IL-7, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, IL-16, IL-17, IL-23, IL-32, M-CSF (CSF-1), IFN-α, IFN-β, IL-1α, IL-1β, IL-1RA, IL-11, IL-17A, IL-17F, IL-19, IL-20, IL-21, IL-22, IL-24, IL-25, IL-26, IL-27, IL-28A, B, IL-29, IL-30, IL-31, IL-33, IL-34, IL-35, IL-36α,β,λ, IL-36Ra, IL-37, TSLP, LIF, OSM, LT-α, LT-β, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, Trail, OPG-L, APRIL, LIGHT, TWEAK, BAFF, TGF-β1, and MIF.

In some embodiments, the target antigen is fused or linked to an immunological fusion partner, comprising CpG ODN (e.g., Class A, B, or C CpG ODNs; non-limiting examples sequences are shown in SEQ ID NO: 108-SEQ ID NO: 119 in which phosphodiester bases are in capital letters, phosphorothioate bases are in lower case letters, and palindromes are underlined and the colon denotes the reflection point), cholera toxin (a non-limiting example sequence is shown in SEQ ID NO: 49), a truncated A subunit coding region derived from a bacterial ADP-ribosylating exotoxin (a non-limiting example sequence is shown in (a non-limiting example sequence is shown in SEQ ID NO: 50), a truncated B subunit coding region derived from a bacterial ADP-ribosylating exotoxin (a non-limiting example sequence is shown in SEQ ID NO: 51), Hp91 (a non-limiting example sequence is shown in SEQ ID NO: 52), CCL20 (a non-limiting example sequence is shown in SEQ ID NO: 53 and SEQ ID NO: 107), CCL3 (a non-limiting example sequence is shown in SEQ ID NO: 54), GM-CSF (a non-limiting example sequence is shown in SEQ ID NO: 55), G-CSF (a non-limiting example sequence is shown in SEQ ID NO: 56), LPS peptide mimic (non-limiting example sequences are shown in SEQ ID NO: 57-SEQ ID NO: 68), shiga toxin (a non-limiting example sequence is shown in SEQ ID NO: 69), diphtheria toxin (a non-limiting example sequence is shown in SEQ ID NO: 70), or CRM₁₉₇ (a non-limiting example sequence is shown in SEQ ID NO: 73).

In some embodiments, the target antigen is fused or linked to an immunological fusion partner, comprising an IL-15 superagonist. Interleukin 15 (IL-15) is a naturally occurring inflammatory cytokine secreted after viral infections. Secreted IL-15 can carry out its function by signaling via its cognate receptor on effector immune cells, and thus, can lead to overall enhancement of effector immune cell activity.

Based on IL-15's broad ability to stimulate and maintain cellular immune responses, it is believed to be a promising immunotherapeutic drug. However, major limitations in clinical development of IL-15 can include low production yields in standard mammalian cell expression systems and short serum half-life. Moreover, the IL-15:IL-15Rα complex, comprising proteins co-expressed by the same cell, rather than the free IL-15 cytokine, can be responsible for stimulating immune effector cells bearing IL-15 βγc receptor.

To contend with these shortcomings, a novel IL-15 superagonist mutant (IL-15N72D) was identified that has increased ability to bind IL-15Rβyc and enhanced biological activity. Addition of either mouse or human IL-15Rα and Fc fusion protein (the Fc region of immunoglobulin) to equal molar concentrations of IL-15N72D can provide a further increase in IL-15 biologic activity, such that IL-15N72D:IL-15Rα/Fc super-agonist complex exhibits a median effective concentration (EC50) for supporting IL-15-dependent cell growth that was greater than 10-fold lower than that of free IL-15 cytokine.

In some embodiments, the IL-15 superagonist can be a novel IL-15 superagonist mutant (IL-15N72D). In certain embodiments, addition of either mouse or human IL-15Rα and Fc fusion protein (the Fc region of immunoglobulin) to equal molar concentrations of IL-15N72D can provide a further increase in IL-15 biologic activity, such that IL-15N72D:IL-15Rα/Fc super-agonist complex exhibits a median effective concentration (EC₅₀) for supporting IL-15-dependent cell growth that can be greater than 10-fold lower than that of free IL-15 cytokine

Thus, in some embodiments, the present disclosure provides a IL-15N72D:IL-15Rα/Fc super-agonist complex with an EC50 for supporting IL-15-dependent cell growth that is greater than 2-fold lower, greater than 3-fold lower, greater than 4-fold lower, greater than 5-fold lower, greater than 6-fold lower, greater than 7-fold lower, greater than 8-fold lower, greater than 9-fold lower, greater than 10-fold lower, greater than 15-fold lower, greater than 20-fold lower, greater than 25-fold lower, greater than 30-fold lower, greater than 35-fold lower, greater than 40-fold lower, greater than 45-fold lower, greater than 50-fold lower, greater than 55-fold lower, greater than 60-fold lower, greater than 65-fold lower, greater than 70-fold lower, greater than 75-fold lower, greater than 80-fold lower, greater than 85-fold lower, greater than 90-fold lower, greater than 95-fold lower, or greater than 100-fold lower than that of free IL-15 cytokine.

In some embodiments, the IL-15 super agonist is a biologically active protein complex of two IL-15N72D molecules and a dimer of soluble IL-15Rα/Fc fusion protein, also known as ALT-803. The composition of ALT-803 and methods of producing and using ALT-803 are described in U.S. Patent Application Publication 2015/0374790, which is herein incorporated by reference. It is known that a soluble IL-15Rα fragment, containing the so-called “sushi” domain at the N terminus (Su), can bear most of the structural elements responsible for high affinity cytokine binding. A soluble fusion protein can be generated by linking the human IL-15RαSu domain (amino acids 1-65 of the mature human IL-15Rα protein) with the human IgG1 CH2-CH3 region containing the Fc domain (232 amino acids). This IL-15RSu/IgG 1 Fc fusion protein can have the advantages of dimer formation through disulfide bonding via IgG1 domains and ease of purification using standard Protein A affinity chromatography methods.

In some embodiments, ALT-803 can have a soluble complex consisting of 2 protein subunits of a human IL-15 variant associated with high affinity to a dimeric IL-15Rα sushi domain/human IgG1 Fc fusion protein. The IL-15 variant is a 114 amino acid polypeptide comprising the mature human IL-15 cytokine sequence with an Asn to Asp substitution at position 72 of helix C N72D). The human IL-15R sushi domain/human IgG1 Fc fusion protein comprises the sushi domain of the IL-15R subunit (amino acids 1-65 of the mature human IL-15Rα protein) linked with the human IgG1 CH2-CH3 region containing the Fc domain (232 amino acids). Aside from the N72D substitution, all of the protein sequences are human. Based on the amino acid sequence of the subunits, the calculated molecular weight of the complex comprising two IL-15N72D polypeptides (an example IL-15N72D sequence is shown in SEQ ID NO: 71) and a disulfide linked homodimeric IL-15RαSu/IgG1 Fc protein (an example IL-15RαSu/Fc domain is shown in SEQ ID NO: 72) is 92.4 kDa. In some embodiments, a recombinant vector encoding for a target antigen and for ALT-803 can have any sequence described herein to encode for the target antigen and can have SEQ ID NO: 71, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 72, in any order, to encode for ALT-803.

Each IL-15N720 polypeptide can have a calculated molecular weight of approximately 12.8 kDa and the IL-15RαSu/IgG 1 Fc fusion protein can have a calculated molecular weight of approximately 33.4 kDa. Both the IL-15N72D and IL-15RαSu/IgG 1 Fc proteins can be glycosylated resulting in an apparent molecular weight of ALT-803 of approximately 114 kDa by size exclusion chromatography. The isoelectric point (pI) determined for ALT-803 can range from approximately 5.6 to 6.5. Thus, the fusion protein can be negatively charged at pH 7.

Combination therapy with Ad5[E1-, E2b-] vectors encoding for a Chikungunya virus antigen and ALT-803 can result in boosting the immune response, such that the combination of both therapeutic moieties acts to synergistically boost the immune response more than either therapy alone. For example, combination therapy with an Ad5[E1-, E2b-] vector encoding for a Chikungunya virus antigen and ALT-803 can result in synergistic enhancement of stimulation of antigen-specific effector CD4+ and CD8+ T cells, stimulation of NK cell response directed towards killing infected cells, stimulation of neutrophils or monocyte cell responses directed towards killing infected cells via antibody dependent cell-mediated cytotoxicity (ADCC), or antibody dependent cellular phagocytosis (ADCP) mechanisms. Combination therapy with an Ad5[E1-, E2b-] vector encoding for a Chikungunya virus antigen and ALT-803 can synergistically boost any one of the above responses, or a combination of the above responses, to vastly improve survival outcomes after administration to a subject in need thereof.

Any of the immunogenicity enhancing agents described herein can be fused or linked to a target antigen by expressing the immunogenicity enhancing agents and the target antigen in the same recombinant vector, using any recombinant vector described herein.

Nucleic acid sequences that encode for such immunogenicity enhancing agents can be any one of SEQ ID NO: 22-SEQ ID NO: 47, SEQ ID NO: 49-SEQ ID NO: 77, and SEQ ID NO: 93-SEQ ID NO: 119 and are summarized in TABLE 1.

TABLE 1 Sequences of Immunological Fusion Partners SEQ ID NO Sequence SEQ ID NO: 22 TAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGL GVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAM ADALNGHFPGDVISVTWQTKSGGTRTGNVTLAEGPPA SEQ ID NO: 23 MHHHHHHTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHI GPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAP INSATAMADALNGHRPGDVISVTWQTKSGGTRTGNVTLAEGPPAEFD DDDKDPPDPHQPDMTKGYCPGGRWGFGDLAVCDGEKYPDGSFWHQ WMQTWFTGPQFYFDCVSGGEPLPGPPPPGGCGGAIPSEQPNAP SEQ ID NO: 24 MHHHHHHTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHI GPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAP INSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAEFP LVPRGSPMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLD FAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQC LSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPS CLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMG QQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMT SQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGV FRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYSGCNKRYFKLSHL QMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCK TCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHN MHQRNMTKLQLAL SEQ ID NO: 25 MHHHHHHTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHI GPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAP INSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAEFI EGRGSGCPLLENVISKTINPQVSKTEYKELLQEFIDDNATTNAIDELKE CFLNQTDETLSNVEVFMQLIYDSSLCDLF SEQ ID NO: 26 MHHHHHHTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHI GPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAP INSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAEF MVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAF QSVVWGLTVGSWIGSSAGLMVAAASPYVAWMSVTAGQAELTAAQV RVAAAAYETAYGLTVPPPVIAENRAELMILIATNLLGQNTPAIAVNEA EYGEMWAQDAAAMFGYAAATATATATLLPFEEAPEMTSAGGLLEQ AAAVEEASDTAAANQLMNNVPQALQQLAQPTQGTTPSSKLGGLWKT VSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAAQ AVQTAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQ AWAAANQAVTPAARALPLTSLTSAAERGPGQMLGGLPVGQMGARA GGGLSGVLRVPPRPYVMPHSPAAGDIAPPALSQDRFADFPALPLDPSA MVAQVGPQVVNINTKLGYNNAVGAGTGIVIDPNGVVLTNNHVIAGA TDINAFSVGSGQTYGVDVVGYDRTQDVAVLQLRGAGGLPSAAIGGG VAVGEPVVAMGNSGGQGGTPRAVPGRVVALGQTVQASDSLTGAEET LNGLIQFDAAIQPGDSGGPVVNGLGQVVGMNTAAS SEQ ID NO: 27 TAASDNFQLSQGGQGFAIPIGQAMAIAGQI SEQ ID NO: 28 TAASDNFQLSQGGQGFAIPIGQAMAIAGQIKLPTVHIGPTAFLGLGVV DNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAMADAL NGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPA SEQ ID NO: 29 TAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGL GVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAM ADALNGHHPGDVISVTWQTKSGGTRTGNVTLAE SEQ ID NO: 30 MSNSRRRSLRWSWLLSVLAAVGLGLATAPAQAAPPALSQDRFADFP ALPLDPSAMVAQVGPQVVNINTKLGYNNAVGAGTGIVIDPNGVVLTN NHVIAGATDINAFSVGSGQTYGVDVVGYDRTQDVAVLQLRGAGGLP SAAIGGGVAVGEPVVAMGNSGGQGGTPRAVPGRVVALGQTVQASDS LTGAEETLNGLIQFDAAIQPGDSGGPVVNGLGQVVGMNTAASDNFQL SQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGLGVVDNNGN GARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAMADALNGHHP GDVISVTWQTKSGGTRTGNVTLAEGPPA SEQ ID NO: 31 MKLKTLALSLLAAGVLAGCSSHSSNMANTQMKSDKIIIAHRGASGYL PEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHELDGLTDVA KKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRF PLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDI AAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLK LVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYAD GVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDAL PAFFTDVNQMYDVLLNKSGATGVFTDFPDTGVEFLKGIK SEQ ID NO: 32 MEINVSKLRTDLPQVGVQPYRQVHAHSTGNPHSTVQNEADYHWRKD PELGFFSHIVGNGCIMQVGPVDNGAWDVGGGWNAETYAAVELIESH STKEEFMTDYRLYIELLRNLADEAGLPKTLDTGSLAGIKTHEYCTNNQ PNNHSDHVDPYPYLAKWGISREQFKHDIENGLTIETGWQKNDTGYW YVHSDGSYPKDKFEKINGTWYYFDSSGYMLADRWRKHTDGNWYWF DNSGEMATGWKIGADKWYYFNEEGAMKTGWVKYKDTWYYLDAK EGAMVSNAFIQSADGTGWYYLKPDGTLADRPEFRMSQMA SEQ ID NO: 33 MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVAD NGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETI KEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAE LSPAAKTGKRKRSQMLFRGRRASQ SEQ ID NO: 34 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFC LLHEGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQA EGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEP IYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL SEQ ID NO: 35 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGI NNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSIISTLT SEQ ID NO: 36 MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFI KELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAE NS SEQ ID NO: 37 MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPR DLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSAT RLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPP LGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGP QDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPEN PPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYR LQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLN QTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVG QDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASA HPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAP SLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVA YTVQVRADTAWLRGVWSQPQRFSIEVQVSDWLIFFASLGSFLSILLVG VLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEA SLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRC KAKM SEQ ID NO: 38 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVI RNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGM AVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDN KMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED SEQ ID NO: 39 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLL DSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNS TGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSL KEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH SEQ ID NO: 40 MSRLPVLLLLQLLVRPGLQAPMTQTTSLKTSWVNCSNMIDEIITHLKQ PPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESIL KNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQT TLSLAIF SEQ ID NO: 41 MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCT ELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQF HRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIM REKYSKCSS SEQ ID NO: 42 MRMLLHLSLLALGAAYVYAIPTEIPTSALVKETLALLSTHRTLLIANET LRIPVPVHKNHQLCTEEIFQGIGTLESQTVQGGTVERLFKNLSLIKKYI DGQKKKCGEERRRVNQFLDYLQEFLGVMNTEWIIES SEQ ID NO: 43 MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLT SSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMA EKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQ MSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTT HLILRSFKEFLQSSLRALRQM SEQ ID NO: 44 MVLTSALLLCSVAGQGCPTLAGILDINFLINKMQEDPASKCHCSANVT SCLCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSVEVLKN NKCPYFSCEQPCNQTTAGNALTFLKSLLEIFQKEKMRGMRGKI SEQ ID NO: 45 MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDA FSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV MPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQ VKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN SEQ ID NO: 46 MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCN GSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSA GQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFNRNFESIIICRDRT SEQ ID NO: 47 MDFQVQIFSFLLISASVIMSRANWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSS NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 49 MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIYTLNDKIFSYT ESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYL TEAKVEKLCVWNNKTPHAIAAISMAN SEQ ID NO: 50 MVKIIFVFFIFLSSFSYANDDKLYRADSRPPDEIKQSGGLMPRGQNEYF DRGTQMNINLYDHARGTQTGFVRHDDGYVSTSISLRSAHLVGQTILS GHSTYYTYVIATAPNMFNVNDVLGAYSPHPDEQEVSALGGIPYSQIYG WYRVHFGVLDEQLHRNRGYRDRYYSNLDIAPAADGYGLAGFPPEHR AWREEPWIHHAPPGCGNAPRSSMSNTCDEKTQSLGVKFLDEYQSKV KRQIFSGYQSDIDTHNRIKDEL SEQ ID NO: 51 MIKLKFGVFFTVLLSSAYAHGTPQNITDLCAEYHNTQIHTLNDKILSYT ESLAGNREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAY LTEAKVEKLCVWNNKTPHAIAAISMAN SEQ ID NO: 52 DPNAPKRPPSAFFLFCSE SEQ ID NO: 53 MCCTKSLLLAALMSVLLLHLCGESEAASNFDCCLGYTDRILHPKFIVG FTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYIVRLLSKKVKN M SEQ ID NO: 54 MQVSTAALAVLLCTMALCNQFSASLAADTPTACCFSYTSRQIPQNFIA DYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYVSDLELSA SEQ ID NO: 55 MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRD TAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLT MMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE SEQ ID NO: 56 MAGPATQSPMKLMALQLLLWHSALWTVQEATPLGPASSLPQSFLLK CLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSLGIPWAPLSSC PSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVAD FATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQ SFLEVSYRVLRHLAQP SEQ ID NO: 57 QEINSSY SEQ ID NO: 58 SHPRLSA SEQ ID NO: 59 SMPNPMV SEQ ID NO: 60 GLQQVLL SEQ ID NO: 61 HELSVLL SEQ ID NO: 62 YAPQRLP SEQ ID NO: 63 TPRTLPT SEQ ID NO: 64 APVHSSI SEQ ID NO: 65 APPHALS SEQ ID NO: 66 TFSNRFI SEQ ID NO: 67 VVPTPPY SEQ ID NO: 68 ELAPDSP SEQ ID NO: 69 TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITG MTVTIKQNACHNGGGFSEVIFR SEQ ID NO: 70 MSRKLFASILIGALLGIGAPPSAHAGADDVVDSSKSFVMENFSSYHGT KPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDN ENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLME QVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI NFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRD KTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPE LSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSI LPGIGSVMGIADGAVHFINTEEIVAQSIALSSLMVAQAIPLVGELVDIGF AAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVE DSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVN GRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIH SNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS SEQ ID NO: 71 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ VISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS SEQ ID NO: 72 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLN KATNVAHWTTPSLKCIREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 73 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD DDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVL ALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPF AEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQAC AGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESP NKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAA WAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEI VAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNR PAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTP LPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKS PVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKV NSKLSLFFEIKS SEQ ID NO: 74 MESHSRAGKSRKSAKFRSISRSLMLCNAKTSDDGSSPDEKYPDPFEISL AQGKEGIFHSSVQLADTSEAGPSSVPDLALASEAAQLQAAGNDRGKT CRRIFFMKESSTASSREKPGKLEAQSSNFLFPKACHQRARSNSTSVNPY CTREIDFPMTKKSAAPTDRQPYSLCSNRKSLSQQLDCPAGKAAGTSRP TRSLSTAQLVQPSGGLQASVISNIVLMKGQAKGLGFSIVGGKDSIYGPI GIYVKTIFAGGAAAADGRLQEGDEILELNGESMAGLTHQDALQKFKQ AKKGLLTLTVRTRLTAPPSLCSHLSPPLCRSLSSSTCITKDSSSFALESPS APISTAKPNYRIMVEVSLQKEAGVGLGIGLCSVPYFQCISGIFVHTLSP GSVAHLDGRLRCGDEIVEISDSPVHCLTLNEVYTILSRCDPGPVPIIVSR HPDPQVSEQQLKEAVAQAVENTKFGKERHQWSLEGVKRLESSWHGR PTLEKEREKNSAPPHRRAQKVMIRSSSDSSYMSGSPGGSPGSGSAEKP SSDVDISTHSPSLPLAREPVVLSIASSRLPQESPPLPESRDSHPPLRLKKS FEILVRKPMSSKPKPPPRKYFKSDSDPQKSLEERENSSCSSGHTPPTCG QEARELLPLLLPQEDTAGRSPSASAGCPGPGIGPQTKSSTEGEPGWRR ASPVTQTSPIKHPLLKRQARMDYSFDTTAEDPWVRISDCIKNLFSPIMS ENHGIEMPLQPNASLNEEEGTQGHPDGTPPKLDTANGTPKVYKSADSS TVKKGPPVAPKPAWFRQSLKGLRNRASDPRGLPDPALSTQPAPASRE HLGSHIRASSSSSSIRQRISSFETFGSSQLPDKGAQRLSLQPSSGEAAKP LGKHEEGRFSGLLGRGAAPTLVPQQPEQVLSSGSPAASEARDPGVSES PPPGRQPNQKTLPPGPDPLLRLLSTQAEESQGPVLKMPSQRARSFPLTR SQSCETKLLDEKTSKLYSISSQVSSAVMKSLLCLPSSISCAQTPCIPKEG ASPTSSSNEDSAANGSAETSALDTGFSLNLSELREYTEGLTEAKEDDD GDHSSLQSGQSVISLLSSEELKKLIEEVKVLDEATLKQLDGIHVTILHK EEGAGLGFSLAGGADLENKVITVHRVFPNGLASQEGTIQKGNEVLSIN GKSLKGTTHHDALAILRQAREPRQAVIVTRKLTPEAMPDLNSSTDSAA SASAASDVSVESTEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTI NRIFKGAASEQSETVQPGDEILQLGGTAMQGLTRFEAWNIIKALPDGP VTIVIRRKSLQSKETTAAGDS SEQ ID NO: 75 MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNL NIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRH LGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCT CVTPIVHHVA SEQ ID NO: 76 RAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETT NDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPS LLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLR FKILRSLQAFVAVAARVFAHGAATLSPIWELKKDVYVVELDWYPDAP GEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYT CHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNY SGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDN KEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDII KPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGK SKREKKDRVFTDKTSATVKRKNASISVRAQDRYYSSSWSEWASVPC S SEQ ID NO: 77 MCFPKVLSDDMKKLKARMVMLLPTSAQGLGAWVSACDTEDTVGHL GPWRDKDPALWCQLCLSSQHQAIERFYDKMQNAESGRGQVMSSLAE LEDDFKEGYLETVAAYYEEQHPELTPLLEKERDGLRCRGNRSPVPDV EDPATEEPGESFCDKVMRWFQAMLQRLQTWWHGVLAWVKEKVVA LVHAVQALWKQFQSFCCSLSELFMSSFQSYGAPRGDKEELTPQKCSE PQSSK SEQ ID NO: 93 GACTACGTTGGTGTAGAAAAATCCTGCCGCCCGGACCCTTAAGGC TGGGACAATTTCTGATAGCTACCCCGACACAGGAGGTTACGGGAT GAGCAATTCGCGCCGCCGCTCACTCAGGTGGTCATGGTTGCTGAGC GTGCTGGCTGCCGTCGGGCTGGGCCTGGCCACGGCGCCGGCCCAG GCGGCCCCGCCGGCCTTGTCGCAGGACCGGTTCGCCGACTTCCCCG CGCTGCCCCTCGACCCGTCCGCGATGGTCGCCCAAGTGGGGCCAC AGGTGGTCAACATCAACACCAAACTGGGCTACAACAACGCCGTGG GCGCCGGGACCGGCATCGTCATCGATCCCAACGGTGTCGTGCTGA CCAACAACCACGTGATCGCGGGCGCCACCGACATCAATGCGTTCA GCGTCGGCTCCGGCCAAACCTACGGCGTCGATGTGGTCGGGTATG ACCGCACCCAGGATGTCGCGGTGCTGCAGCTGCGCGGTGCCGGTG GCCTGCCGTCGGCGGCGATCGGTGGCGGCGTCGCGGTTGGTGAGC CCGTCGTCGCGATGGGCAACAGCGGTGGGCAGGGCGGAACGCCCC GTGCGGTGCCTGGCAGGGTGGTCGCGCTCGGCCAAACCGTGCAGG CGTCGGATTCGCTGACCGGTGCCGAAGAGACATTGAACGGGTTGA TCCAGTTCGATGCCGCGATCCAGCCCGGTGATTCGGGCGGGCCCGT CGTCAACGGCCTAGGACAGGTGGTCGGTATGAACACGGCCGCGTC CGATAACTTCCAGCTGTCCCAGGGTGGGCAGGGATTCGCCATTCCG ATCGGGCAGGCGATGGCGATCGCGGGCCAGATCCGATCGGGTGGG GGGTCACCCACCGTTCATATCGGGCCTACCGCCTTCCTCGGCTTGG GTGTTGTCGACAACAACGGCAACGGCGCACGAGTCCAACGCGTGG TCGGGAGCGCTCCGGCGGCAAGTCTCGGCATCTCCACCGGCGACG TGATCACCGCGGTCGACGGCGCTCCGATCAACTCGGCCACCGCGA TGGCGGACGCGCTTAACGGGCATCATCCCGGTGACGTCATCTCGGT GACCTGGCAAACCAAGTCGGGCGGCACGCGTACAGGGAACGTGAC ATTGGCCGAGGGACCCCCGGCCTGATTTCGTCGCGGATACCACCC GCCGGCCGGCCAATTGGATTGGCGCCAGCCGTGATTGCCGCGTGA GCCCCCGAGTTCCGTCTCCCGTGCGCGTGGCATCGTGGAAGCAATG AACGAGGCAGAACACAGCGTCGAGCACCCTCCCGTGCAGGGCAGT CACGTCGAAGGCGGTGTGGTCGAGCATCCGGATGCCAAGGACTTC GGCAGCGCCGCCGCCCTGCCCGCCGATCCGACCTGGTTTAAGCAC GCCGTCTTCTACGAGGTGCTGGTCCGGGCGTTCTTCGACGCCAGCG CGGACGGTTCCGGCGATCTGCGTGGACTCATCGATCGCCTCGACTA CCTGCAGTGGCTTGGCATCGACTGCATCTGGTTGCCGCCGTTCTAC GACTCGCCGCTGCGCGACGGCGGTTACGACATTCGCGACTTCTACA AGGTGCTGCCCGAATTCGGCACCGTCGACGATTTCGTCGCCCTGGT CGACGCCGCTCACCGGCGAGGTATCCGCATCATCACCGACCTGGT GATGAATCACACCTCGGAGTCGCACCCCTGGTTTCAGGAGTCCCGC CGCGACCCAGACGGACCGTACGGTGACTATTACGTGTGGAGCGAC ACCAGCGAGCGCTACACCGACGCCCGGATCATCTTCGTCGACACC GAAGAGTCGAACTGGTCATTCGATCCTGTCCGCCGACAGTTCTACT GGCACCGATTCTT SEQ ID NO: 94 ACGGCCGCGTCCGATAACTTCCAGCTGTCCCAGGGTGGGCAGGGA TTCGCCATTCCGATCGGGCAGGCGATGGCGATCGCGGGCCAGATC CGATCGGGTGGGGGGTCACCCACCGTTCATATCGGGCCTACCGCCT TCCTCGGCTTGGGTGTTGTCGACAACAACGGCAACGGCGCACGAG TCCAACGCGTGGTCGGGAGCGCTCCGGCGGCAAGTCTCGGCATCT CCACCGGCGACGTGATCACCGCGGTCGACGGCGCTCCGATCAACT CGGCCACCGCGATGGCGGACGCGCTTAACGGGCATCATCCCGGTG ACGTCATCTCGGTGACCTGGCAAACCAAGTCGGGCGGCACGCGTA CAGGGAACGTGACATTGGCCGAGGGACCCCCGGCC SEQ ID NO: 95 CATATGCATCACCATCACCATCACACGGCCGCGTCCGATAACTTCC AGCTGTCCCAGGGTGGGCAGGGATTCGCCATTCCGATCGGGCAGG CGATGGCGATCGCGGGCCAGATCCGATCGGGTGGGGGGTCACCCA CCGTTCATATCGGGCCTACCGCCTTCCTCGGCTTGGGTGTTGTCGA CAACAACGGCAACGGCGCACGAGTCCAACGCGTGGTCGGGAGCGC TCCGGCGGCAAGTCTCGGCATCTCCACCGGCGACGTGATCACCGC GGTCGACGGCGCTCCGATCAACTCGGCCACCGCGATGGCGGACGC GCTTAACGGGCATCATCCCGGTGACGTCATCTCGGTGACCTGGCAA ACCAAGTCGGGCGGCACGCGTACAGGGAACGTGACATTGGCCGAG GGACCCCCGGCCGAATTCGACGACGACGACAAGGATCCACCTGAC CCGCATCAGCCGGACATGACGAAAGGCTATTGCCCGGGTGGCCGA TGGGGTTTTGGCGACTTGGCCGTGTGCGACGGCGAGAAGTACCCC GACGGCTCGTTTTGGCACCAGTGGATGCAAACGTGGTTTACCGGCC CACAGTTTTACTTCGATTGTGTCAGCGGCGGTGAGCCCCTCCCCGG CCCGCCGCCACCGGGTGGTTGCGGTGGGGCAATTCCGTCCGAGCA GCCCAACGCTCCCTGAGAATTC SEQ ID NO: 96 CATATGCATCACCATCACCATCACACGGCCGCGTCCGATAACTTCC AGCTGTCCCAGGGTGGGCAGGGATTCGCCATTCCGATCGGGCAGG CGATGGCGATCGCGGGCCAGATCCGATCGGGTGGGGGGTCACCCA CCGTTCATATCGGGCCTACCGCCTTCCTCGGCTTGGGTGTTGTCGA CAACAACGGCAACGGCGCACGAGTCCAACGCGTGGTCGGGAGCGC TCCGGCGGCAAGTCTCGGCATCTCCACCGGCGACGTGATCACCGC GGTCGACGGCGCTCCGATCAACTCGGCCACCGCGATGGCGGACGC GCTTAACGGGCATCATCCCGGTGACGTCATCTCGGTGACCTGGCAA ACCAAGTCGGGCGGCACGCGTACAGGGAACGTGACATTGGCCGAG GGACCCCCGGCCGAATTCCCGCTGGTGCCGCGCGGCAGCCCGATG GGCTCCGACGTTCGGGACCTGAACGCACTGCTGCCGGCAGTTCCGT CCCTGGGTGGTGGTGGTGGTTGCGCACTGCCGGTTAGCGGTGCAG CACAGTGGGCTCCGGTTCTGGACTTCGCACCGCCGGGTGCATCCGC ATACGGTTCCCTGGGTGGTCCGGCACCGCCGCCGGCACCGCCGCC GCCGCCGCCGCCGCCGCCGCACTCCTTCATCAAACAGGAACCGAG CTGGGGTGGTGCAGAACCGCACGAAGAACAGTGCCTGAGCGCATT CACCGTTCACTTCTCCGGCCAGTTCACTGGCACAGCCGGAGCCTGT CGCTACGGGCCCTTCGGTCCTCCTCCGCCCAGCCAGGCGTCATCCG GCCAGGCCAGGATGTTTCCTAACGCGCCCTACCTGCCCAGCTGCCT CGAGAGCCAGCCCGCTATTCGCAATCAGGGTTACAGCACGGTCAC CTTCGACGGGACGCCCAGCTACGGTCACACGCCCTCGCACCATGC GGCGCAGTTCCCCAACCACTCATTCAAGCATGAGGATCCCATGGG CCAGCAGGGCTCGCTGGGTGAGCAGCAGTACTCGGTGCCGCCCCC GGTCTATGGCTGCCACACCCCCACCGACAGCTGCACCGGCAGCCA GGCTTTGCTGCTGAGGACGCCCTACAGCAGTGACAATTTATACCAA ATGACATCCCAGCTTGAATGCATGACCTGGAATCAGATGAACTTA GGAGCCACCTTAAAGGGCCACAGCACAGGGTACGAGAGCGATAA CCACACAACGCCCATCCTCTGCGGAGCCCAATACAGAATACACAC GCACGGTGTCTTCAGAGGCATTCAGGATGTGCGACGTGTGCCTGG AGTAGCCCCGACTCTTGTACGGTCGGCATCTGAGACCAGTGAGAA ACGCCCCTTCATGTGTGCTTACTCAGGCTGCAATAAGAGATATTTT AAGCTGTCCCACTTACAGATGCACAGCAGGAAGCACACTGGTGAG AAACCATACCAGTGTGACTTCAAGGACTGTGAACGAAGGTTTTTTC GTTCAGACCAGCTCAAAAGACACCAAAGGAGACATACAGGTGTGA AACCATTCCAGTGTAAAACTTGTCAGCGAAAGTTCTCCCGGTCCGA CCACCTGAAGACCCACACCAGGACTCATACAGGTGAAAAGCCCTT CAGCTGTCGGTGGCCAAGTTGTCAGAAAAAGTTTGCCCGGTCAGA TGAATTAGTCCGCCATCACAACATGCATCAGAGAAACATGACCAA ACTCCAGCTGGCGCTTTGAGAATTC SEQ ID NO: 97 CATATGCATCACCATCACCATCACACGGCCGCGTCCGATAACTTCC AGCTGTCCCAGGGTGGGCAGGGATTCGCCATTCCGATCGGGCAGG CGATGGCGATCGCGGGCCAGATCCGATCGGGTGGGGGGTCACCCA CCGTTCATATCGGGCCTACCGCCTTCCTCGGCTTGGGTGTTGTCGA CAACAACGGCAACGGCGCACGAGTCCAACGCGTGGTCGGGAGCGC TCCGGCGGCAAGTCTCGGCATCTCCACCGGCGACGTGATCACCGC GGTCGACGGCGCTCCGATCAACTCGGCCACCGCGATGGCGGACGC GCTTAACGGGCATCATCCCGGTGACGTCATCTCGGTGACCTGGCAA ACCAAGTCGGGCGGCACGCGTACAGGGAACGTGACATTGGCCGAG GGACCCCCGGCCGAATTCATCGAGGGAAGGGGCTCTGGCTGCCCC TTATTGGAGAATGTGATTTCCAAGACAATCAATCCACAAGTGTCTA AGACTGAATACAAAGAACTTCTTCAAGAGTTCATAGACGACAATG CCACTACAAATGCCATAGATGAATTGAAGGAATGTTTTCTTAACCA AACGGATGAAACTCTGAGCAATGTTGAGGTGTTTATGCAATTAAT ATATGACAGCAGTCTTTGTGATTTATTTTAAGAATTC SEQ ID NO: 98 ATGCATCACCATCACCATCACACGGCCGCGTCCGATAACTTCCAGC TGTCCCAGGGTGGGCAGGGATTCGCCATTCCGATCGGGCAGGCGA TGGCGATCGCGGGCCAGATCCGATCGGGTGGGGGGTCACCCACCG TTCATATCGGGCCTACCGCCTTCCTCGGCTTGGGTGTTGTCGACAA CAACGGCAACGGCGCACGAGTCCAACGCGTGGTCGGGAGCGCTCC GGCGGCAAGTCTCGGCATCTCCACCGGCGACGTGATCACCGCGGT CGACGGCGCTCCGATCAACTCGGCCACCGCGATGGCGGACGCGCT TAACGGGCATCATCCCGGTGACGTCATCTCGGTGACCTGGCAAAC CAAGTCGGGCGGCACGCGTACAGGGAACGTGACATTGGCCGAGGG ACCCCCGGCCGAATTCATGGTGGATTTCGGGGCGTTACCACCGGA GATCAACTCCGCGAGGATGTACGCCGGCCCGGGTTCGGCCTCGCT GGTGGCCGCGGCTCAGATGTGGGACAGCGTGGCGAGTGACCTGTT TTCGGCCGCGTCGGCGTTTCAGTCGGTGGTCTGGGGTCTGACGGTG GGGTCGTGGATAGGTTCGTCGGCGGGTCTGATGGTGGCGGCGGCC TCGCCGTATGTGGCGTGGATGAGCGTCACCGCGGGGCAGGCCGAG CTGACCGCCGCCCAGGTCCGGGTTGCTGCGGCGGCCTACGAGACG GCGTATGGGCTGACGGTGCCCCCGCCGGTGATCGCCGAGAACCGT GCTGAACTGATGATTCTGATAGCGACCAACCTCTTGGGGCAAAAC ACCCCGGCGATCGCGGTCAACGAGGCCGAATACGGCGAGATGTGG GCCCAAGACGCCGCCGCGATGTTTGGCTACGCCGCGGCGACGGCG ACGGCGACGGCGACGTTGCTGCCGTTCGAGGAGGCGCCGGAGATG ACCAGCGCGGGTGGGCTCCTCGAGCAGGCCGCCGCGGTCGAGGAG GCCTCCGACACCGCCGCGGCGAACCAGTTGATGAACAATGTGCCC CAGGCGCTGCAACAGCTGGCCCAGCCCACGCAGGGCACCACGCCT TCTTCCAAGCTGGGTGGCCTGTGGAAGACGGTCTCGCCGCATCGGT CGCCGATCAGCAACATGGTGTCGATGGCCAACAACCACATGTCGA TGACCAACTCGGGTGTGTCGATGACCAACACCTTGAGCTCGATGTT GAAGGGCTTTGCTCCGGCGGCGGCCGCCCAGGCCGTGCAAACCGC GGCGCAAAACGGGGTCCGGGCGATGAGCTCGCTGGGCAGCTCGCT GGGTTCTTCGGGTCTGGGCGGTGGGGTGGCCGCCAACTTGGGTCG GGCGGCCTCGGTCGGTTCGTTGTCGGTGCCGCAGGCCTGGGCCGC GGCCAACCAGGCAGTCACCCCGGCGGCGCGGGCGCTGCCGCTGAC CAGCCTGACCAGCGCCGCGGAAAGAGGGCCCGGGCAGATGCTGG GCGGGCTGCCGGTGGGGCAGATGGGCGCCAGGGCCGGTGGTGGGC TCAGTGGTGTGCTGCGTGTTCCGCCGCGACCCTATGTGATGCCGCA TTCTCCGGCAGCCGGCGATATCGCCCCGCCGGCCTTGTCGCAGGAC CGGTTCGCCGACTTCCCCGCGCTGCCCCTCGACCCGTCCGCGATGG TCGCCCAAGTGGGGCCACAGGTGGTCAACATCAACACCAAACTGG GCTACAACAACGCCGTGGGCGCCGGGACCGGCATCGTCATCGATC CCAACGGTGTCGTGCTGACCAACAACCACGTGATCGCGGGCGCCA CCGACATCAATGCGTTCAGCGTCGGCTCCGGCCAAACCTACGGCG TCGATGTGGTCGGGTATGACCGCACCCAGGATGTCGCGGTGCTGC AGCTGCGCGGTGCCGGTGGCCTGCCGTCGGCGGCGATCGGTGGCG GCGTCGCGGTTGGTGAGCCCGTCGTCGCGATGGGCAACAGCGGTG GGCAGGGCGGAACGCCCCGTGCGGTGCCTGGCAGGGTGGTCGCGC TCGGCCAAACCGTGCAGGCGTCGGATTCGCTGACCGGTGCCGAAG AGACATTGAACGGGTTGATCCAGTTCGATGCCGCGATCCAGCCCG GTGATTCGGGCGGGCCCGTCGTCAACGGCCTAGGACAGGTGGTCG GTATGAACACGGCCGCGTCCTAGG SEQ ID NO: 107 mcctkslllaalmsvlllhlcgeseasnfdcclgytdrilhpkfivgftrqlanegcdinaiifht kkklsvcanpkqtwvkyivrllskkvknm SEQ ID NO: 108 ggGGTCAACGTTGAgggggg SEQ ID NO: 109 ggGGGACGA:TCGTCgggggg SEQ ID NO: 110 gggGACGAC:GTCGTGgggggg SEQ ID NO: 111 tccatgacgttcctgatgct SEQ ID NO: 112 tccatgacgttcctgacgtt SEQ ID NO: 113 tcgtcgttttgtcgttttgtcgtt SEQ ID NO: 114 tcg tcg ttg tcg ttt tgt cgt t SEQ ID NO: 115 tcg acg ttc gtc gtt cgt cgt tc SEQ ID NO: 116 tcg cga cgt tcg ccc gac gtt cgg ta SEQ ID NO: 117 tcgtcgttttcggcgc:gcgccg SEQ ID NO: 118 tcgtcgtcgttc:gaacgacgttgat SEQ ID NO: 119 tcg cga acg ttc gcc gcg ttc gaa cgc gg

TABLE 2 Tools to construct Mycobacterium sp.-Derived Ra12 Sequences SEQ ID NO Sequence SEQ ID NO: 99 CAATTACATATGCATCACCATCACCATCACACGGCCGCGTCCGATA ACTTC SEQ ID NO: 100 CTAATCGAATTCGGCCGGGGGTCCCTCGGCCAA SEQ ID NO: 101 CAATTAGAATTCGACGACGACGACAAGGATCCACCTGACCCGCAT CAG SEQ ID NO: 102 CAATTAGAATTCTCAGGGAGCGTTGGGCTGCTC SEQ ID NO: 103 GCGAAGCTTATGAAGTTGCTGATGGTCCTCATGC SEQ ID NO: 104 CGGCTCGAGTTAAAATAAATCACAAAGACTGCTGTC SEQ ID NO: 105 MHHHHHH SEQ ID NO: 106 DDDK

In some embodiments, the nucleic acid sequences for the target antigen and the immunological fusion partner are not separated by any nucleic acids. In other embodiments, a nucleic acid sequence that encodes for a linker can be inserted between the nucleic acid sequence encoding for any target antigen described herein and the nucleic acid sequence encoding for any immunological fusion partner described herein. Thus, in certain embodiments, the protein produced following immunization with the viral vector containing a target antigen, a linker, and an immunological fusion partner can be a fusion protein comprising the target antigen of interest followed by the linker and ending with the immunological fusion partner, thus linking the target antigen to an immunological fusion partner that increases the immunogenicity of the target antigen of interest via a linker. In some embodiments, the sequence of linker nucleic acids can be from about 1 to about 150 nucleic acids long, from about 5 to about 100 nucleic acids along, or from about 10 to about 50 nucleic acids in length. In some embodiments, the nucleic acid sequences can encode one or more amino acid residues. In some embodiments, the amino acid sequence of the linker can be from about 1 to about 50, or about 5 to about 25 amino acid residues in length. In some embodiments, the sequence of the linker comprises less than 10 amino acids. In some embodiments, the linker can be a polyalanine linker, a polyglycine linker, or a linker with both alanines and glycines.

Nucleic acid sequences that encode for such linkers can be any one of SEQ ID NO: 78-SEQ ID NO: 92 and are summarized in TABLE 3.

TABLE 3 Sequences of Linkers SEQ ID NO Sequence SEQ ID NO: 78 MAVPMQLSCSR SEQ ID NO: 79 RSTG SEQ ID NO: 80 TR SEQ ID NO: 81 RSQ SEQ ID NO: 82 RSAGE SEQ ID NO: 83 RS SEQ ID NO: 84 GG SEQ ID NO: 85 GSGGSGGSG SEQ ID NO: 86 GGSGGSGGSGG SEQ ID NO: 87 GGSGGSGGSGGSGG SEQ ID NO: 88 GGSGGSGGSGGSGGSGG SEQ ID NO: 89 GGSGGSGGSGGSGGSGGSGG SEQ ID NO: 90 GGSGGSGGSGGSGGSGGSGGSGG SEQ ID NO: 91 GGSGGSGGSGGSGGSG SEQ ID NO: 92 GSGGSGGSGGSGGSGG

VI. Methods of Use

The adenovirus vectors can be used in a number of vaccine settings for generating an immune response against one or more alphavirus target antigens as described herein, especially chikungunya virus target antigens. The adenovirus vectors are of particular importance because of the finding that they can be used to generate immune responses in subjects who have preexisting immunity to adenovirus or adenovirus vectors and can be used in vaccination regimens that include multiple rounds of immunization using the adenovirus vectors, regimens not possible using previous generation adenovirus vectors.

Generally, generating an immune response can comprise an induction of a humoral response and/or a cell-mediated response. In certain embodiments, it is desirable to increase an immune response against a target antigen of interest. As such “generating an immune response” or “inducing an immune response” can comprise any statistically significant change, e.g., increase in the number of one or more immune cells (T cells, B cells, antigen-presenting cells, dendritic cells, neutrophils, and the like) or in the activity of one or more of these immune cells (CTL activity, HTL activity, cytokine secretion, change in profile of cytokine secretion, etc.).

The skilled artisan would readily appreciate that a number of methods for establishing whether an alteration in the immune response has taken place are available. A variety of methods for detecting alterations in an immune response (e.g., cell numbers, cytokine expression, cell activity) are known in the art and are useful in the context of the instant invention. Illustrative methods are described in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001 John Wiley & Sons, NY, NY) Ausubel et al. (2001 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, NY); Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.) and elsewhere. Illustrative methods useful in this context can include intracellular cytokine staining (ICS), ELISpot, proliferation assays, cytotoxic T cell assays including chromium release or equivalent assays, and gene expression analysis using any number of polymerase chain reaction (PCR) or RT-PCR based assays.

In certain embodiments, generating an immune response comprises an increase in target antigen-specific CTL activity of about 1.5 to 20 or more fold, at least, about, or at most 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or any range or number derived therefrom in a subject administered the adenovirus vectors as compared to a control. In another embodiment, generating an immune response comprises an increase in target-specific CTL activity of about 1.5 to 20, or more fold in a subject administered the adenovirus vectors as compared to a control. In a further embodiment, generating an immune response that comprises an increase in target antigen-specific cell mediated immunity activity as measured by ELISpot assays measuring cytokine secretion, such as interferon-gamma (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), granzyme, or other cytokines, of about 1.5 to 20, or more fold as compared to a control.

In a further embodiment, generating an immune response comprises an increase in target-specific antibody production of between 1.5 and 5 fold in a subject administered the adenovirus vectors as compared to an appropriate control. In another embodiment, generating an immune response comprises an increase in target-specific antibody production of about 1.5 to 20, or more fold in a subject administered the adenovirus vector as compared to a control.

Thus, certain aspects can provide methods for generating an immune response against an alphavirus target antigen of interest comprising administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigen; and readministering the adenovirus vector at least once to the subject; thereby generating an immune response against the target antigen. Certain other aspects can provide methods for generating an immune response against alphavirus target antigens of interest comprising administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigens; and readministering the adenovirus vector at least once to the subject; thereby generating an immune response against the target antigens. In certain embodiments, there can be provided methods wherein the vector administered is not a gutted vector.

In further embodiments, methods can be provided for generating an immune response against an alphavirus virus target antigen in a subject, wherein the subject has pre-existing immunity to Ad, by administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigen; and re-administering the adenovirus vector at least once to the subject; thereby generating an immune response against the alphavirus virus target antigen. In still further embodiments, methods can be provided for generating an immune response against alphavirus virus target antigens in a subject, wherein the individual has pre-existing immunity to Ad, by administering to the subject an adenovirus vector comprising: a) a replication defective adenovirus vector, wherein the adenovirus vector has a deletion in the E2b region, and b) nucleic acids encoding the target antigens; and re-administering the adenovirus vector at least once to the subject; thereby generating an immune response against the alphavirus virus target antigens.

With regard to preexisting immunity to Ad, this can be determined using methods known in the art, such as antibody-based assays to test for the presence of Ad antibodies. Further, in certain embodiments, the methods can include first determining that an individual has preexisting immunity to Ad then administering the E2b deleted adenovirus vectors as described herein.

In certain aspects, there can be provided methods of generating an immune response against an alphavirus target antigen, such as those described elsewhere herein.

In particular aspects, there are provided methods of generating an immune response against an alphavirus, such as those described elsewhere herein.

VII. Pharmaceutical Compositions

As noted elsewhere herein, the adenovirus vector can comprise nucleic acid sequences that encode one or more target antigens of interest from any one or more of the infectious agents against which an immune response is to be generated. For example, a target antigen can include, but is not limited to, viral antigen protein, such as E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, and nsP4.

For administration, the adenovirus vector stock can be combined with an appropriate buffer, physiologically acceptable carrier, excipient or the like. In certain embodiments, an appropriate number of adenovirus vector particles are administered in an appropriate buffer, such as, sterile PBS.

In certain circumstances it can be desirable to deliver the adenovirus vector composition disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. In other embodiments, a E2b deleted adenovirus vector can be delivered in pill form, delivered by swallowing or by suppository.

Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Pat. No. 5,466,468). The form can be sterile and can be fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms, such as bacteria, molds and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it can include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions can be especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15^(th) Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations can need to meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biology standards.

The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” can refer to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.

Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and from disease to disease, and can be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines can be administered by injection (e.g., intracutaneous, intraperitoneal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), in pill form (e.g., swallowing, suppository for vaginal or rectal delivery). In certain embodiments, between 1 and 3 doses can be administered over a 6 week period and further booster vaccinations can be given periodically thereafter.

In various embodiments, the replication defective adenovirus is administered at a dose that suitable for effecting an immune response as described herein. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10⁸ virus particles to about 5×10¹³ virus particles per immunization. In some cases, the replication defective adenovirus is administered at a dose that is from about 1×10⁹ to about 5×10¹² virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10⁸ virus particles to about 5×10⁸ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5×10⁸ virus particles to about 1×10⁹ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10⁹ virus particles to about 5×10⁹ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5×10⁹ virus particles to about 1×10¹⁰ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹⁰ virus particles to about 5×10¹⁰ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5×10¹⁰ virus particles to about 1×10¹¹ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹¹ virus particles to about 5×10¹¹ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5×10¹¹ virus particles to about 1×10¹² virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹² virus particles to about 5×10¹² virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 5×10¹² virus particles to about 1×10¹³ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹³ virus particles to about 5×10¹³ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10⁸ virus particles to about 5×10¹⁰ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹⁰ virus particles to about 5×10¹² virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹¹ virus particles to about 5×10¹³ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10⁸ virus particles to about 1×10¹⁰ virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹⁰ virus particles to about 1×10¹² virus particles per immunization. In some embodiments, the replication defective adenovirus is administered at a dose from about 1×10¹¹ virus particles to about 5×10¹³ virus particles per immunization. In some cases, the replication defective adenovirus is administered at a dose that is greater than or equal to 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.5×10¹², 2×10¹², 3×10¹², or more virus particles (VP) per immunization. In some cases, the replication defective adenovirus is administered at a dose that is less than or equal to 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰, 6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹, 6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 1.5×10¹², 2×10¹², 3×10¹², or more virus particles per immunization. In various embodiments, a desired dose described herein is administered in a suitable volume of formulation buffer, for example a volume of about 0.1-10 mL, 0.2-8 mL, 0.3-7 mL, 0.4-6 mL, 0.5-5 mL, 0.6-4 mL, 0.7-3 mL, 0.8-2 mL, 0.9-1.5 mL, 0.95-1.2 mL, or 1.0-1.1 mL. Those of skill in the art appreciate that the volume can fall within any range bounded by any of these values (e.g., about 0.5 mL to about 1.1 mL).

A suitable dose can be an amount of an adenovirus vector that, when administered as described above, is capable of promoting a target antigen immune response as described elsewhere herein. In certain embodiments, the immune response is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the target antigen antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing alphavirus infected cells in vitro, or other methods known in the art for monitoring immune responses.

In general, an appropriate dosage regimen provides the adenovirus vectors in an amount sufficient to provide prophylactic benefit. Protective immune responses can generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which can be performed using samples obtained from a patient before and after immunization (vaccination).

While one advantage is the capability to administer multiple vaccinations with the same adenovirus vectors, particularly in individuals with preexisting immunity to Ad, the adenovirus vaccines can also be administered as part of a prime and boost regimen. A mixed modality priming and booster inoculation scheme can result in an enhanced immune response.

Thus, one aspect is a method of priming a subject with a plasmid vaccine, such as a plasmid vector comprising a target antigen of interest, by administering the plasmid vaccine at least one time, allowing a predetermined length of time to pass, and then boosting by administering the adenovirus vector. Multiple primings, e.g., 1-3, can be employed, although more can be used. The length of time between priming and boost can vary from about six months to a year, but other time frames can be used.

VIII. Kits

A composition, immunotherapy, or vaccine described herein can be supplied in the form of a kit. The kits of the present disclosure can further comprise instructions regarding the dosage and/or administration regimen information.

In some embodiments, a kit comprises a composition and method for providing a vaccine as described herein. In some embodiments kits can further comprise components useful in administering the kit components and instructions on how to prepare the components. In some embodiments, the kit can further comprise software for conducting monitoring of patients before and after vaccination with appropriate laboratory tests, or communicating results and patient data with medical staff.

The components of the kit can be in dry or liquid form. If they are in dry form, the kit can include a solution to solubilize the dried material. The kit can also include transfer factor in liquid or dry form. In some embodiments, if the transfer factor is in dry form, the kit includes a solution to solubilize the transfer factor. The kit can also include containers for mixing and preparing the components. The kit can also include instrument for assisting with the administration such as, for example, needles, tubing, applicator, inhalant, syringe, pipette, forceps, measured spoon, eye dropper, or any such medically approved delivery vehicle. The kits or drug delivery systems as described herein also can include a means for containing compositions of the present disclosure in close confinement for commercial sale and distribution.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

EXAMPLES

The following examples are included to further describe some aspects of the present disclosure, and should not be used to limit the scope of the present disclosure.

Example 1 Production of Single-targeted Ad5 [E1-, E2b-]-CHIKV Vaccine

This example illustrates the results of construction and expression of Ad5 [E1-, E2b-] vectors containing a CHIKV antigen.

Ad5 [E1-, E2b-]-CHIKV vaccine is an adenovirus serotype 5 (Ad5) vector that was modified by removal of E1, E2b, and E3 gene regions, and insertion of a CHIKV gene.

In this Example, the nucleic acid sequence encoding a CHIKV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 2) including CHIKV structural proteins C, E3, E2, 6K, and E1 was cloned into an Ad5 [E1-, E2b-]-based platform to produce Ad5 [E1-, E2b-]-CHIKV_(str) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-CHIKV_(str) was produced in E.C7 cells (FIG. 2). The replication-deficient virus was propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 62ittered. Viral infectious titer was determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration was determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

Infection of E.C7 cells with Ad5 [E1-, E2b-]-CHIKV_(str) resulted in expression of CHIKV structural proteins. CHIKV structural protein expression was detectable by western blot with an anti-CHIKV antibody (FIG. 3).

Example 2 Multiple Injections of an Ad5 [E1-, E2b-]-CHIKV_(str) Generated Protective Immunity Against CHIKV-Infection

This example illustrates the results of injection of an Ad5 [E1-, E2b-]-CHIKV_(str) for generating protective immunity against CHIKV infection.

Groups of ten (10) mice each were immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VPs Ad5 [E1-, E2b-]-CHIKV_(str). Control mice were injected with PBS. Mice were then challenged with a lethal dose of CHIKV by injection of the virus into the footpad. Control mice succumbed to infection within 7 days (FIG. 4A), lost weight (FIG. 4B), and demonstrated prolonged inflammation at the footpad (FIG. 4C). By contrast, vaccinated mice survived CHIKV infection (FIG. 4A), did not lose weight (FIG. 4B), and demonstrated rapid resolution of inflammation of the footpad (FIG. 4C).

Example 3 Production of a Multi-Targeted Ad5 [E1-, E2b-]-CHIKV Antigen Insert

This example illustrates construction of an Ad5 [E1-, E2b-] vector containing multiple CHIKV antigens.

To produce the Ad5 [E1-, E2b-] containing multiple CHIKV genes, three individual alphavirus antigen gene sequences ((1) C, E3, E2, 6K, and E1 gene ((nucleotides 7567-11313 of SEQ ID NO: 1); (2) NS gene 1; (3) NS gene 2) are separated by “self-cleaving” 2A peptide derived from Porcine teschovirus-1 and Thosea asigna virus respectively (FIG. 5A) (de Felipe P and Ryan M Traffic 2004 5(8), 616-26; Holst J et al. Nature Immunol. 2008 9:658-66; Kim J H et al. PloS One, 2011 6(4), e18556. Doi:10.1371/journal.pone.0018556). As the 2A peptides are translated on the ribosome, the peptide bond between the final two residues of the 2A peptide is never formed resulting in distinctly expressed proteins in one ribosomal pass (FIG. 5B). The use of two 2A peptide sequences separating the three genes are found in near stoichiometric expression of the three proteins (FIG. 5B).

Example 4 Production and Testing of a Single-Targeted Ad5 [E1-, E2b-]-ONNV Vaccine

This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing an ONNV antigen, and testing for expression and protective immunity by multiple injections of the vectors.

Ad5 [E1-, E2b-]-ONNV_(str) vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of E1, E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding an ONNV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 5) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-ONNV_(str) is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and is 63ittered. Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

ONNV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1-, E2b-]-ONNV_(str). ONNV structural protein expression is detected by western blot with an anti-ONNV antibody.

Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VPs Ad5 [E1-, E2b-]-ONNV_(str). Control mice are injected with PBS or 10⁹ VPs Ad5-null. Mice are then challenged with a lethal dose of ONNV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.

Example 5 Production and Testing of a Single-Targeted Ad5 [E1-, E2b-]-MAYV Vaccine

This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing an MAYV antigen and testing for expression and protective immunity by multiple injection of the vectors.

Ad5 [E1-, E2b-]-MAYV_(str) vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of E1, E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding an MAYV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 8) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-MAYV_(str) is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 64ittered. Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

MAYV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1-, E2b-]-MAYV_(str). MAYV structural protein expression is detected by western blot with an anti-MAYV antibody.

Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VP Ad5 [E1-, E2b-]-MAYV_(str). Control mice are injected with PBS or 10⁹ VPs Ad5-null. Mice are then challenged with a lethal dose of MAYV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.

Example 6 Production and Testing of a Single-Targeted Ad5 [E1-, E2b-]-RRV Vaccine

This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing a RRV antigen and testing for expression and protective immunity by multiple injection of the vectors.

Ad5 [E1-, E2b-]-RRV_(str) vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of E1, E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding a RRV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 11) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-RRV_(str) is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 65ittered. Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

RRV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1-, E2b-]-RRV_(str). RRV structural protein expression is detected by western blot with an anti-RRV antibody.

Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VP Ad5 [E1-, E2b-]-RRV_(str). Control mice are injected with PBS or 10⁹ VPs Ad5-null. Mice are then challenged with a lethal dose of RRV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.

Example 7 Production and Testing of a Single-Targeted Ad5 [E1-, E2b-]-VEEV Vaccine

This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing a VEEV antigen and testing for expression and protective immunity by multiple injection of the vectors.

Ad5 [E1-, E2b-]-VEEV_(str) vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of E1, E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding a VEEV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 14) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-VEEV_(str) is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 65ittered. Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

VEEV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1-, E2b-]-VEEV_(str). VEEV structural protein expression is detected by western blot with an anti-VEEV antibody.

Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VP Ad5 [E1-, E2b-]-VEEV_(str). Control mice are injected with PBS or 10⁹ VPs Ad5-null. Mice are then challenged with a lethal dose of VEEV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.

Example 8 Production and Testing of a Single-Targeted Ad5 [E1-, E2b-]-EEEV Vaccine

This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing an EEEV antigen and testing for expression and protective immunity by multiple injection of the vectors.

Ad5 [E1-, E2b-]-EEEV_(str) vaccine is an adenovirus serotype 5 (Ad5) vector that is modified by removal of E1, E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding an EEEV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 17) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-EEEV_(str) is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 66ittered. Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

EEEV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1-, E2b-]-EEEV_(str). EEEV structural protein expression is detected by western blot with an anti-EEEV antibody.

Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VP Ad5 [E1-, E2b-]-EEEV_(str). Control mice are injected with PBS or 10⁹ VPs Ad5-null. Mice are then challenged with a lethal dose of EEEV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.

Example 9 Production and Testing of a Single-Targeted Ad5 [E1-, E2b-]-WEEV Vaccine

This example illustrates the construction of Ad5 [E1-, E2b-] vectors containing a WEEV antigen and testing for expression and protective immunity by multiple injection of the vectors.

Ad5 [E1-, E2b-]-WEEV_(str) vaccine is an adenovirus serotype 5 (Ad5) vector that has been modified by removal of E1, E2b, and E3 gene regions, and insertion of the nucleic acid sequence encoding a WEEV structural polyprotein (the amino acid sequence is set forth in SEQ ID NO: 20) using a homologous recombination-based approach.

Ad5 [E1-, E2b-]-WEEV_(str) is produced in E.C7 cells. The replication-deficient virus is propagated in the E.C7 packaging cell line, purified by ultracentrifugation or ion exchange column purification, and 66ittered. Viral infectious titer is determined as plaque-forming units (PFUs) on an E.C7 cell monolayer. The VP concentration is determined by sodium dodecyl sulfate (SDS) disruption and spectrophotometry at 260 nm and 280 nm.

WEEV structural proteins are expressed as a result of infection of E.C7 cells with Ad5 [E1-, E2b-]-WEEV_(str). WEEV structural protein expression is detected by western blot with an anti-WEEV antibody.

Groups of ten (10) mice each are immunized two times subcutaneously at weekly intervals with a dose of 10⁹ VPs Ad5 [E1-, E2b-]-WEEV_(str). Control mice are injected with PBS or 10⁹ VPs Ad5-null. Mice are then challenged with a lethal dose of WEEV by injection of the virus. The survival rate, immune response, and weight are compared between the mice injected with the vaccine and the control group.

Example 10 Pre-Clinical Studies of Ad5 [E1-, E2b-]-Chikungunya Vaccines in Mice

This example illustrates pre-clinical studies of Ad5 [E1-, E2b-]-Chikungunya vaccines in mice, including assessment of cell mediate immune (CMI) responses, cytolytic T lymphocyte (CTL) responses, intracellular cytokine expression, and antibody secretion. Pre-clinical studies included administration of Ad5 [E1-, E2b-]-Chikungunya vaccines, a comparison to controls, and an assessment of the immune responses in mice.

Pre-Clinical Assessment of Ad5 [E1-, E2b-]-Chikungunya Vaccines

CMI and CTL Responses.

CMI and CTL responses in mice were evaluated by an enzyme-linked immunospot (ELISPOT) assay. FIG. 6 illustrates CMI responses (IFN-γ) and CTL responses and Granzyme B responses in splenocytes from immunized or control mice using an ELISpot assay. C57BL/6 mice were immunized two times at two-week intervals with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with 1×10⁰ VPs Ad5 [E1-, E2b-]-null (empty vector controls). One week after the final immunization, splenocytes from individual mice were tested for induction of immune responses after exposure of cells to Chikungunya virus peptides. The data show the cumulative number of spot forming cells (SFCs) per 10⁶ splenocytes after exposure to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays—CHIKV peptide pool 1 comprised peptides 1-103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-3 10). Additional splenocytes were separately exposed to an SIV-nef peptide pools as a negative control prior to assay measurements. FIG. 6A illustrates CMI responses in Ad5 [E1-, E2b-]-CHIK immunized and control C57BL/6 mice as measured by IFN-γ secreting SFCs using an ELISpot assay. Specificity of the response is evidenced by the lack of reactivity of splenocytes to the negative control SIV-nef peptide pool. A high number of IFN-γ secreting SFCs were induced in Ad5 [E1-, E2b-]-CHIK immunized mice as compared to control mice. FIG. 6B illustrates CTL responses in Ad5 [E1-, E2b-]-CHIK immunized and control C57BL/6 mice as measured by Granzyme B (Gr-B) secreting SFCs using an ELISpot assay. Specificity of the response is evidenced by the lack of reactivity of splenocytes to the negative control SIV-nef peptide pool. A high number of Granzyme B secreting SFCs were induced in Ad5 [E1-, E2b-]-CHIK immunized mice as compared to control mice.

Intracellular Cytokine Expression.

Flow cytometry analysis revealed the levels of lymphocyte activation as measured by evaluating intracellular cytokine expression. FIG. 7 illustrates lymphocyte activation in splenocytes from immunized or control C57BL/6 mice as measured by intracellular expression of IFN-γ or IFN-γ/TNF-α analyzed by flow cytometry. C57BL/6 mice were immunized two times at two-week intervals with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-null (empty vector controls). One week after the final immunization, splenocytes from individual mice were exposed to three separate pools of Chikungunya virus peptides (peptide numbers for Chikungunya were large enough to merit division into three separate pools to use in assays—CHIKV peptide pool 1 comprised peptides 1-103, CHIKV peptide pool 2 comprising peptides 104-207, and peptide pool 3 comprising peptides 208-310) and analyzed by flow cytometry for induction of intracellular cytokine expression. FIG. 7A illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD8+ splenocytes from immunized mice expressed intracellular IFN-γ as compared to negative controls. FIG. 7B illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD4+ splenocytes from immunized mice expressed intracellular IFN-γ as compared to negative controls. FIG. 7C illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ and TNF-α in CD8+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD8+ splenocytes from immunized mice expressed intracellular IFN-γ and TNF-α as compared to negative controls. FIG. 7D illustrates lymphocyte activation as measured by flow cytometry analysis of intracellular expression of IFN-γ and TNF-α in CD4+ splenocytes after exposure of splenocytes from immunized mice and control mice to three separate pools of Chikungunya virus peptides and controls (media and SIV-nef peptide pools). A higher percentage of CD4+ splenocytes from immunized mice expressed intracellular IFN-γ and TNF-α as compared to negative controls.

Antigen-Specific Antibody Production.

Chikungunya-specific IgG antibodies were measured in the serum of immunized mice by an enzyme-linked immunosorbent assay (ELISA). FIG. 8 illustrates anti-Chikungunya envelope protein-2 antibody responses in immunized mice as compared to control mice as measured by a quantitative enzyme-linked immunosorbent assay (ELISA). C57BL/6 mice were immunized two times at two-week intervals with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-CHIK vaccine comprising SEQ ID NO: 1 or with 1×10¹⁰ VPs Ad5 [E1-, E2b-]-null (empty vector controls). One week after the final immunization, sera from mice were evaluated for induction of antibody responses. Blood was collected by cheek pouch laceration under anesthesia. Anti-Chikungunya specific antibody responses were induced at higher levels in immunized mice as compared to control mice.

Example 11 Prevention of Chikungunya Infection with Ad5 [E1-, E2b-]-Chikungunya Vaccine

This example illustrates prevention of Chikungunya infection by prophylaxis with any Ad5 [E1-, E2b-]-Chikungunya vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1-SEQ ID NO: 3) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-Chikungunya vaccine is constructed as described in EXAMPLE 1 for a single-targeted Chikungunya vaccine or is constructed as described in EXAMPLE 3 for a multi-targeted Chikungunya vaccine. The Ad5 [E1-, E2b-]-Chikungunya vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against Chikungunya virus and protection against infection by Chikungunya virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-Chikungunya vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-Chikungunya vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 12 Prevention of Chikungunya Infection with Ad5 [E1-, E2b-]-Chikungunya Vaccine and a Co-Stimulatory Molecule

This example illustrates prevention of Chikungunya infection by prophylaxis with any Ad5 [E1-, E2b-]-Chikungunya vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1-SEQ ID NO: 3) inserted into the adenovirus vector in combination with any co-stimulatory molecule described herein. An Ad5 [E1-, E2b-]-Chikungunya vaccine is constructed as described in EXAMPLE 1 for a single-targeted Chikungunya vaccine or is constructed as described in EXAMPLE 3 for a multi-targeted Chikungunya vaccine. The Ad5 [E1-, E2b-]-Chikungunya vaccine is administered subcutaneously, intradermally, or intramuscularly to a subject once or every two weeks for a total of two immunizations. The Ad vaccine is co-administered with a co-stimulatory molecule, such as a toll-like receptor (TLR) agonist mixed with the vaccine formulation. Cellular and humoral immune responses against Chikungunya virus and protection against infection by Chikungunya virus is induced after immunization of a subject with the combination of Ad5 [E1-, E2b-]-Chikungunya vaccine and co-stimulatory molecule. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-Chikungunya vaccine and co-stimulatory molecule is conferred to the subject.

Example 13 Prevention of Chikungunya Infection with Ad5 [E1-, E2b-]-Chikungunya Vaccine and an Immunological Fusion Partner

This example illustrates prevention of Chikungunya infection by prophylaxis with any Ad5 [E1-, E2b-]-CHIK vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, Chikungunya antigens (e.g., SEQ ID NO: 1-SEQ ID NO: 3) inserted into the adenovirus vector as well as any immunological fusion partner described herein, also encoded by the adenovirus vector. An Ad5 [E1-, E2b-]-CHIK vaccine is constructed as described in EXAMPLE 1 for a single-targeted Chikungunya vaccine or is constructed as described in EXAMPLE 3 for a multi-targeted Chikungunya vaccine with the Ad vector additionally encoding for any immunological fusion partner disclosed herein. The Ad5 [E1-, E2b-]-Chikungunya vaccine is administered subcutaneously, intradermally, or intramuscularly to a subject, once or every two weeks for a total of two immunizations. Vaccines with immunological fusion partners are administered subcutaneously, intradermally, or intramuscularly. Cellular and humoral immune responses against Chikungunya virus and protection against infection by Chikungunya virus is induced after immunization of a subject with Ad5 [E1-, E2b-]-Chikungunya vaccine-immunological fusion partner and co-stimulatory molecule. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-Chikungunya vaccine-immunological fusion partner is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 14 Prevention of O'Nyong Nyong Virus Infection with Ad5 [E1-, E2b-]-O'Nyong Nyong Virus (ONNV) Vaccine

This example illustrates prevention of o'nyong-nyong virus (ONNV) infection by prophylaxis with any Ad5 [E1-, E2b-]-ONNV vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, ONNV antigens (e.g., SEQ ID NO: 4-SEQ ID NO: 6) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-ONNV vaccine is constructed as adapted from EXAMPLE 1 or as described in EXAMPLE 4 for a single-targeted ONNV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted ONNV vaccine. The Ad5 [E1-, E2b-]-ONNV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against ONNV virus and protection against infection by ONNV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-ONNV vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-ONNV vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 15 Prevention of Ross River Virus Infection with Ad5 [E1-, E2b-]-Ross River Virus (RRV) Vaccine

This example illustrates prevention of Ross River virus (RRV) infection by prophylaxis with any Ad5 [E1-, E2b-]-RRV vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, RRV antigens (e.g., SEQ ID NO: 10-SEQ ID NO: 12) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-RRV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 6 for a single-targeted RRV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted RRV vaccine. The Ad5 [E1-, E2b-]-RRV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against RRV virus and protection against infection by RRV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-RRV vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-RRV vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 16 Prevention of Mayaro Fever Virus Infection with Ad5 [E1-, E2b-]-Mayaro Fever Virus (MAYV) Vaccine

This example illustrates prevention of Marayo fever virus (MAYV) infection by prophylaxis with any Ad5 [E1-, E2b-]-MAYV vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, MAYV antigens (e.g., SEQ ID NO: 7-SEQ ID NO: 9) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-MAYV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 5 for a single-targeted MAYV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted MAYV vaccine. The Ad5 [E1-, E2b-]-MAYV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against MAYV virus and protection against infection by MAYV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-MAYV vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-MAYV vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 17 Prevention of Venezuelan Equine Encephalitis Virus Infection with Ad5 [E1-, E2b-]-Venezuelan Equine Encephalitis Virus (VEEV) Vaccine

This example illustrates prevention of Venezuelan equine encephalitis virus (VEEV) infection by prophylaxis with any Ad5 [E1-, E2b-]-VEEV vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, VEEV antigens (e.g., SEQ ID NO: 13-SEQ ID NO: 15) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-VEEV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 7 for a single-targeted VEEV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted VEEV vaccine. The Ad5 [E1-, E2b-]-VEEV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against VEEV virus and protection against infection by VEEV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-VEEV vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-VEEV vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 18 Prevention of Western Equine Encephalomyelitis Virus Infection with Ad5 [E1-, E2b-]-Western Equine Encephalomyelitis Virus (WEEV) Vaccine

This example illustrates prevention of Western equine encephalomyelitis virus (WEEV) infection by prophylaxis with any Ad5 [E1-, E2b-]-WEEV vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, WEEV antigens (e.g., SEQ ID NO: 19-SEQ ID NO: 21) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-WEEV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 9 for a single-targeted WEEV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted WEEV vaccine. The Ad5 [E1-, E2b-]-WEEV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against WEEV virus and protection against infection by WEEV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-WEEV vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-WEEV vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 19 Prevention of Eastern Equine Encephalitis Virus Infection with Ad5 [E1-, E2b-]-Eastern Equine Encephalitis Virus (EEEV) Vaccine

This example illustrates prevention of Eastern equine encephalitis virus (EEEV) infection by prophylaxis with any Ad5 [E1-, E2b-]-EEEV vaccine of this disclosure including an Ad5 [E1-, E2b-] with any one of, or any combination of, EEEV antigens (e.g., SEQ ID NO: 16-SEQ ID NO: 18) inserted into the adenovirus vector. An Ad5 [E1-, E2b-]-EEEV vaccine is constructed as adapted from EXAMPLE 1 or described in EXAMPLE 8 for a single-targeted EEEV vaccine or is constructed as adapted from EXAMPLE 3 for a multi-targeted EEEV vaccine. The Ad5 [E1-, E2b-]-EEEV vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against EEEV virus and protection against infection by EEEV virus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-EEEV vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-EEEV vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

Example 20 Prevention of Alphavirus Infections with Ad5 [E1-, E2b-]-Alphavirus Vaccine

This example illustrates prevention of alphavirus infections by prophylaxis with any Ad5 [E1-, E2b-]-alphavirus vaccine. The Ad5 [E1-, E2b-]-alphavirus vaccine is comprised of any of combination of: single-targeted or multi-targeted Ad5 [E1, E2b]-ONNV vector as described in EXAMPLE 15, single-targeted or multi-targeted Ad5 [E1, E2b]-CHIK vector as described in EXAMPLE 11, single-targeted or multi-targeted Ad5 [E1, E2b]-RRV vector as described in EXAMPLE 16, single-targeted or multi-targeted Ad5 [E1, E2b]-MAYV vector as described in EXAMPLE 17, single-targeted or multi-targeted Ad5 [E1, E2b]-VEEV vector as described in EXAMPLE 18, single-targeted or multi-targeted Ad5 [E1, E2b]-WEEV vector as described in EXAMPLE 19, an single-targeted or multi-targeted Ad5 [E1, E2b]-RRV vector as described in EXAMPLE 20. Alternatively, the Ad5 [E1-, E2b-]-alphavirus vaccine is comprised of an Ad5 [E1-, E2b-] with any combination of at least two antigens from different alphaviruses inserted into the adenovirus vector. For example, the at least two antigens is comprised of any combination of an ONNV antigen (e.g., any one of SEQ ID NO: 4-SEQ ID NO: 6), a CHIK antigen (e.g., any one of SEQ ID NO: 1-SEQ ID NO: 3), an EEEV antigen (e.g., any one of SEQ ID NO: 16-SEQ ID NO: 18), a WEEV antigen (e.g., any one of SEQ ID NO: 19-SEQ ID NO: 21), a VEEV antigen (e.g., any one of SEQ ID NO: 13-SEQ ID NO: 15), a MAYV antigen (e.g., any one of SEQ ID NO: 7-SEQ ID NO: 9), and/or a RRV antigen (e.g., any one of SEQ ID NO: 10-SEQ ID NO: 12). The Ad5 [E1-, E2b-]-alphavirus vaccine is administered to a subject subcutaneously, intradermally, or intramuscularly, once or every two weeks for a total of two immunizations. Cellular and humoral immune responses against alphaviruses and protection against infection by alphavirus is induced after immunization of a subject with the Ad5 [E1-, E2b-]-alphavirus vaccine. In other words, immunity by prophylaxis with the Ad5 [E1-, E2b-]-alphavirus vaccine is conferred to the subject. The subject is any animal including a human, a non-human primate, or any other non-human animal.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

SEQUENCES SEQ ID NO Sequence SEQ ID NO: 1 ATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTAC TCTGCAAAGCAAGAGATTAAGAACCCATCATGGATCCTGTGTACGT GGACATAGACGCTGACAGCGCCTTTTTGAAGGCCCTGCAACGTGCG TACCCCATGTTTGAGGTGGAACCTAGGCAGGTCACACCGAATGACC ATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGA GCAGGAAATTGATCCCGACTCAACCATCCTGGATATTGGTAGTGCG CCAGCAAGGAGGATGATGTCGGACAGGAAGTACCACTGCGTTTGCC CGATGCGCAGTGCAGAAGATCCCGAGAGACTCGCCAATTATGCGA GAAAGCTAGCATCTGCCGCAGGAAAAGTCCTGGACAGAAACATCT CTGGAAAGATCGGGGACTTACAAGCAGTAATGGCCGTGCCAGACA CGGAGACGCCAACATTCTGCTTACACACAGATGTATCATGTAGACA GAGAGCAGACGTCGCGATATACCAAGACGTCTATGCTGTACACGCA CCCACGTCGCTATACCACCAGGCGATTAAAGGGGTCCGATTGGCGT ACTGGGTAGGGTTTGACACAACCCCGTTCATGTACAATGCCATGGC GGGTGCCTACCCCTCATACTCGACAAATTGGGCAGATGAGCAGGTA CTGAAGGCTAAGAACATAGGATTATGTTCAACAGACCTGACGGAA GGTAGACGAGGCAAATTGTCTATTATGAGAGGAAAAAAGCTAGAA CCGTGCGACCGTGTGCTGTTCTCAGTAGGGTCAACGCTCTACCCGG AAAGCCGTAAGCTACTTAAGAGCTGGCACCTACCATCGGTGTTCCA TTTAAAGGGCAAGCTCAGCTTCACATGCCGCTGTGATACAGTGGTT TCGTGCGAAGGCTACGTCGTTAAGAGAATAACGATGAGCCCAGGCC TTTACGGAAAAACCACAGGGTATGCGGTAACCCACCACGCAGACG GATTCCTGATGTGCAAGACCACCGACACGGTTGACGGCGAAAGAGT GTCATTCTCGGTGTGCACGTACGTGCCGGCGACCATTTGTGATCAA ATGACCGGCATCCTTGCTACAGAAGTCACGCCGGAGGATGCACAGA AGCTGTTGGTGGGGCTGAACCAGAGAATAGTGGTTAACGGCAGAA CGCAACGGAATACGAACACCATGAAAAACTATATGATTCCCGTGGT CGCCCAAGCCTTCAGTAAGTGGGCAAAGGAGTGCCGGAAAGACAT GGAAGATGAAAAACTCCTGGGGGTCAGAGAAAGAACACTGACCTG CTGCTGTCTATGGGCATTTAAGAAGCAGAAAACACACACGGTCTAC AAGAGGCCTGATACCCAGTCAATTCAGAAGGTTCAGGCCGAGTTTG ACAGCTTTGTGGTACCGAGCCTGTGGTCGTCCGGGTTGTCAATCCC GTTGAGGACTAGAATCAAATGGTTGTTAAGCAAGGTGCCAAAAACC GACCTGACCCCATACAGCGGGGACGCCCAAGAAGCCCGGGACGCA GAAAAAGAAGCAGAGGAAGAACGAGAAGCAGAACTGACTCTTGAA GCCCTACCACCCCTTCAGGCAGCACAGGAAGATGTTCAGGTCGAAA TCGACGTGGAACAGCTTGAGGACAGAGCGGGTGCAGGAATAATAG AGACTCCGAGAGGAGCTATCAAAGTTACTGCCCAACCAACAGACC ACGTCGTGGGAGAGTACTTGGTTCTTTCCCCGCAGACCGTACTACG TAGCCAAAAGCTTAGCCTGATTCACGCTTTGGCGGAGCAAGTGAAG ACGTGCACGCACAGCGGACGAGCAGGGAGGTATGCGGTCGAAGCG TACGACGGCAGAGTCCTAGTGCCCTCAGGCTACGCAATCTCGCCTG AAGACTTCCAGAGCCTAAGCGAAAGCGCAACGATGGTGTACAACG AAAGAGAGTTCGTAAACAGAAAGCTACACCATATTGCGATGCATG GACCAGCCCTGAACACCGACGAAGAGTCGTATGAGCTGGTGAGGG CAGAGAGGACAGAACACGAGTACGTCTACGACGTGGACCAGAGAA GATGCTGTAAGAAGGAAGAAGCTGCAGGACTGGTACTGGTGGGCG ACTTGACTAATCCGCCCTACCACGAATTCGCATATGAAGGGCTAAA AATCCGCCCTGCCTGCCCATACAAAATTGCAGTCATAGGAGTCTTC GGAGTACCAGGATCTGGCAAGTCAGCTATTATCAAGAACCTAGTTA CCAGGCAAGACCTGGTGACTAGCGGAAAGAAAGAAAACTGCCAAG AAATCACCACCGACGTGATGAGACAGAGAGGTCTAGAGATATCTG CACGTACGGTTGACTCGCTGCTCTTGAATGGATGTAACAGACCAGT CGACGTGTTGTACGTAGACGAGGCGTTTGCGTGCCACTCTGGAACG TTACTTGCATTGATCGCCTTGGTGAGACCAAGACAGAAAGTTGTAC TTTGTGGTGACCCGAAGCAGTGCGGCTTCTTCAATATGATGCAGAT GAAAGTCAACTATAATCACAACATCTGCACCCAAGTGTACCACAAA AGTATCTCCAGGCGGTGTACACTGCCTGTGACTGCCATTGTGTCATC GTTGCATTACGAAGGCAAAATGCGCACTACGAATGAGTACAACAA GCCGATTGTAGTGGACACTACAGGCTCAACAAAACCTGACCCTGGA GATCTCGTGTTAACGTGCTTCAGAGGATGGGTTAAACAACTGCAAA TTGACTATCGTGGACACGAGGTCATGACAGCAGCCGCATCCCAAGG GTTAACCAGAAAAGGAGTTTACGCAGTTAGGCAAAAAGTTAACGA AAACCCGCTTTATGCATCAACGTCAGAGCACGTCAACGTACTCCTA ACGCGTACGGAAGGTAAACTGGTATGGAAGACACTCTCCGGTGACC CGTGGATAAAGACGCTGCAGAACCCACCGAAAGGAAACTTCAAAG CAACTATTAAGGAGTGGGAGGTGGAGCATGCATCAATAATGGCGG GCATCTGCAGTCACCAAATGACCTTTGATACATTCCAAAACAAAGC CAACGTTTGTTGGGCTAAGAGTTTGGTCCCTATCCTCGAAACAGCG GGGATAAAACTAAACGACAGGCAGTGGTCCCAGATAATTCAAGCC TTCAAAGAAGACAAAGCATATTCACCCGAAGTAGCCCTGAATGAA ATATGCACGCGCATGTATGGGGTGGATCTAGACAGCGGGCTATTTT CTAAACCGTTGGTGTCTGTGTATTACGCGGATAACCACTGGGATAA TAGGCCTGGAGGGAAGATGTTCGGATTCAACCCCGAGGCAGCATCC ATTCTAGAAAGAAAGTATCCATTTACAAAAGGGAAGTGGAACATC AACAAGCAGATCTGCGTGACTACCAGGAGGATAGAAGACTTCAAC CCTACCACCAACATTATACCGGCCAACAGGAGACTACCACACTCAT TAGTGGCCGAACACCGCCCAGTAAAAGGGGAAAGAATGGAATGGC TGGTTAACAAGATAAACGGCCACCACGTGCTCCTGGTCAGTGGCTG TAGCCTTGCACTGCCTACTAAGAGAGTCACTTGGGTAGCGCCACTA GGTGTCCGCGGAGCGGACTATACATACAACCTAGAGTTGGGTCTGC CAGCAACGCTTGGTAGGTATGACCTAGTGGTCATAAACATCCACAC ACCTTTTCGCATACACCATTATCAACAGTGCGTAGACCACGCAATG AAACTGCAAATGCTCGGGGGTGACTCATTGAGACTGCTCAAACCGG GTGGCTCTCTATTGATCAGAGCATATGGTTACGCAGATAGAACCAG TGAACGAGTCATCTGCGTATTGGGACGCAAGTTTAGATCATCTAGA GCGTTGAAACCACCATGTGTCACCAGCAACACTGAGATGTTTTTTCT ATTCAGCAACTTTGACAATGGCAGAAGGAATTTCACAACTCATGTC ATGAACAATCAACTGAATGCAGCCTTTGTAGGACAGGCCACCCGAG CAGGATGTGCACCGTCGTACCGGGTAAAACGCATGGATATCGCGAA GAACGATGAAGAGTGCGTAGTCAACGCCGCCAACCCTCGCGGGTTA CCAGGTGACGGTGTTTGCAAGGCAGTATACAAAAAATGGCCGGAG TCCTTTAAGAACAGTGCAACACCAGTGGGAACCGCAAAAACAGTC ATGTGCGGTACGTATCCAGTAATCCACGCCGTTGGACCAAACTTCT CTAATTATTCGGAGTCTGAAGGGGACCGAGAATTGGCGGCTGCCTA TCGAGAAGTCGCAAAGGAGGTAACTAGACTGGGAGTAAATAGTGT AGCTATACCTCTCCTCTCCACAGGTGTATACTCAGGAGGGAAAGAC AGGCTGACCCAGTCACTGAACCACCTCTTTACAGCCATGGACTCGA CGGATGCAGACGTGGTCATCTACTGCCGCGACAAAGAATGGGAGA AGAAAATATCTGAGGCCATACAGATGCGGACCCAAGTGGAGCTGC TGGATGAGCACATCTCCATAGACTGCGATGTTGTTCGCGTGCACCC TGACAGCAGCTTGGCAGGCAGAAAAGGATACAGCACCACGGAAGG CGCACTGTACTCATATCTAGAAGGGACCCGTTTTCACCAAACGGCA GTGGATATGGCAGAGATATATACTATGTGGCCAAAGCAAACAGAG GCCAACGAGCAAGTTTGCCTATATGCCCTGGGGGAAAGTATTGAAT CGATCAGGCAGAAATGCCCGGTGGATGATGCAGATGCATCATCTCC CCCGAAAACTGTCCCGTGCCTCTGCCGTTACGCCATGACACCAGAA CGCGTTACCCGACTTCGCATGAACCATGTCACAAGCATAATTGTGT GTTCTTCGTTTCCCCTTCCAAAGTACAAAATAGAAGGAGTGCAAAA AGTCAAATGCTCCAAGGTAATGCTATTTGACCACAACGTGCCATCG CGCGTAAGTCCAAGGGAATACAGACCTTCCCAGGAGTCTGTACAGG AAGCGAGTACGACCACGTCACTGACGCATAGCCAATTCGATCTAAG CGTTGACGGCAAGATACTGCCCGTCCCGTCAGACCTGGATGCTGAC GCCCCAGCCCTAGAACCAGCCCTTGACGACGGGGCGATACACACGT TGCCATCTGCAACCGGAAACCTTGCGGCCGTGTCTGACTGGGTAAT GAGCACCGTACCTGTCGCGCCGCCCAGAAGAAGGCGAGGGAGAAA CCTGACTGTGACATGCGACGAGAGAGAAGGGAATATAACACCCAT GGCTAGCGTCCGATTCTTTAGGGCAGAGCTGTGTCCAGTCGTACAA GAAACAGCGGAGACGCGTGACACAGCTATGTCTCTTCAGGCACCGC CGAGTACCGCCACGGAACTGAGTCACCCGCCGATCTCCTTCGGTGC ACCAAGCGAGACGTTCCCCATCACATTTGGGGACTTCAACGAAGGA GAAATCGAAAGCTTGTCTTCTGAGCTACTAACTTTCGGAGACTTCCT ACCCGGAGAAGTGGATGATTTGACAGATAGCGACTGGTCCACGTGC TCAGACACGGACGACGAGTTACGACTAGACAGGGCAGGTGGGTAT ATATTCTCGTCGGACACTGGTCCAGGTCATTTACAACAGAAGTCAG TACGCCAGTCAGTGCTGCCGGTGAACACCCTGGAGGAAGTCCACGA GGAGAAGTGTTACCCACCTAAGCTGGATGAAGCAAAGGAGCAACT ACTACTTAAGAAACTCCAGGAGAGTGCATCCATGGCCAACAGAAG CAGGTATCAGTCGCGCAAAGTAGAAAACATGAAAGCAACAATCAT CCAGAGACTAAAGAGAGGCTGTAGATTATACTTAATGTCAGAGACC CCAAAAGTCCCTACCTACCGGACCACATATCCGGCGCCTGTGTACT CGCCTCCGATTAACGTCCGACTGTCCAACCCCGAGTCCGCAGTGGC AGCATGCAATGAGTTCTTGGCTAGAAACTATCCAACTGTTTCATCAT ACCAAATCACCGACGAGTATGATGCATATCTAGACATGGTGGACGG GTCGGAGAGTTGTCTGGACCGAGCGACATTCAATCCGTCAAAACTT AGGAGCTACCCAAAACAGCACGCTTACCACGCGCCCTCCATCAGAA GCGCTGTACCGTCCCCATTCCAGAACACACTACAGAATGTACTGGC AGCAGCCACGAAAAGAAACTGCAACGTCACACAGATGAGGGAATT ACCCACTTTGGACTCAGCAGTATTCAACGTGGAGTGTTTCAAAAAA TTCGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCCAGCCCTA TCAGGATAACAACTGAGAATTTAACAACCTATGTTACTAAACTAAA GGGGCCAAAAGCAGCAGCGCTATTTGCAAAAACCCATAATCTGCTG CCACTGCAGGAAGTGCCAATGGATAGGTTCACAGTAGACATGAAA AGGGATGTGAAGGTGACTCCTGGTACAAAGCACACAGAGGAAAGA CCTAAGGTACAGGTTATACAGGCGGCTGAACCCTTGGCAACAGCAT ACCTATGTGGGATTCACAGAGAGCTGGTTAGGAGGCTGAACGCCGT CCTCCTACCCAATGTACATACACTATTTGACATGTCTGCCGAGGATT TCGATGCCATCATAGCCGCACACTTTAAGCCAGGAGACACTGTTTT AGAAACGGACATAGCCTCCTTTGATAAGAGCCAAGATGATTCACTT GCGCTTACTGCTTTAATGCTGTTAGAGGATTTAGGGGTGGATCACTC CCTGTTGGACTTGATAGAGGCTGCTTTCGGAGAGATTTCCAGCTGTC ATCTACCGACAGGTACGCGCTTCAAGTTCGGCGCCATGATGAAATC TGGTATGTTCCTAACTCTGTTCGTCAACACACTGCTAAATATCACCA TCGCCAGCCGAGTGCTGGAAGATCGTCTGACAAAATCCGCGTGCGC AGCCTTCATCGGCGACGACAACATAATACATGGAGTCGTCTCCGAT GAATTGATGGCAGCCAGATGCGCCACTTGGATGAACATGGAAGTG AAGATCATAGATGCAGTTGTATCCCAGAAAGCCCCTTACTTTTGTG GAGGGTTTATACTGCACGATATCGTGACAGGAACAGCTTGCAGAGT GGCAGACCCGCTAAAAAGGCTATTTAAACTGGGCAAACCGCTAGC GGCAGGTGACGAACAAGATGAGGATAGAAGACGAGCGCTGGCTGA CGAAGTGGTCAGATGGCAACGAACAGGGCTAATTGATGAGTTGGA GAAAGCGGTATACTCTAGGTATGAAGTGCAGGGTATATCAGTTGTG GTAATGTCCATGGCCACCTTTGCAAGCTCCAGATCCAACTTCGAGA AGCTCAGAGGACCCGTCGTAACTTTGTACGGCGGTCCTAAATAGGT ACGCACTACAGCTACCTATTTTGCAGAAGCCGACAGTAAGTACCTA AACACTAATCAGCTACAATGGAGTTCATCCCAACCCAAACTTTTTA CAACAGGAGGTACCAGCCTCGACCCTGGACTCCGCGCCCTACTATC CAAGTCATCAGGCCCAGACCGCGCCCGCAGAGGCAAGCTGGGCAA CTTGCCCAGCTGATCTCAGCAGTTAATAAACTGACAATGCGCGCGG TACCCCAACAGAAGCCACGCAAGAATCGGAAGAATAAGAAGCAAA AGCAAAAGCAGCAGGCGCCACAAAACAACACAAACCAAAAGAAG CAGCCACCTAAAAAGAAACCAGCTCAAAAGAAAAAGAAGCCGGGC CGCAGAGAGAGGATGTGCATGAAAATCGAAAATGACTGTATTTTCG AAGTCAAGCACGAAGGTAAGGTAACAGGTTACGCGTGCTTGGTGG GGGACAAAGTAATGAAACCAGCACACGTAAAGGGGACCATCGATA ACGCGGACCTGGCCAAATTGGCCTTTAAGCGGTCATCTAAGTACGA CCTTGAATGCGCGCAGATACCCGTGCACATGAAGTCCGACGCTTCG AAGTTCACCCATGAGAAACCGGAGGGGTACTACAACTGGCACCAC GGAGCAGTACAGTACTCAGGAGGCCGGTTCACCATCCCTACAGGTG CGGGCAAACCAGGGGACAGCGGTAGACCGATCTTCGACAACAAGG GACGCGTGGTGGCCATAGTCTTAGGAGGAGCTAATGAAGGAGCCC GTACAGCCCTCTCAGTGGTGACCTGGAATAAAGACATTGTCACTAA AATCACCCCTGAGGGAGCCGAAGAGTGGAGTCTTGCCATCCCAGTT ATGTGCCTGTTGGCAAATACCACGTTCCCCTGCTCCCAGCCCCCTTG CATACCCTGCTGCTACGAAAAGGAACCGGAGGAAACCCTACGCAT GCTTGAGGACAACGTCATGAGACCTGGGTACTATCAGCTGCTACAA GCATCATTAACATGTTCTCCCCACCGCCAGCGACGCAGCACCAAGG ACAACTTCAATGTCTATAAAGCCACAAGACCATACCTAGCTCACTG TCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCACTA GAACGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAG GTCTCCTTGCAAATTGGAATAGGGACGGATGATAGCCATGATTGGA CCAAGCTGCGTTACATGGACAATCACATACCAGCAGACGCAGGGA GGGCCGGGCTATTTGTAAGAACATCAGCACCATGCACGATTACTGG AACAATGGGACACTTCATCCTGGCCCGATGTCCGAAAGGAGAAACT CTGACGGTGGGATTCACTGACAGTAGGAAGATTAGTCACTCATGTA CGCACCCATTTCACCACGACCCTCCTGTGATAGGCCGGGAAAAATT CCATTCCCGACCGCAGCACGGTAAAGAGCTACCTTGCAGCACGTAC GTGCAGAGCAACGCCGCAACTGCCGAGGAGATAGAGGTACACATG CCCCCAGACACCCCTGATCGCACATTGCTGTCACAACAGTCCGGCA ACGTAAAGATCACAGTCAATAGTCAGACGGTGCGGTATAAGTGTAA TTGCGGTGGCTCAAATGAAGGACTAATAACTACAGATAAAGTGATT AATAACTGCAAGGTTGATCAATGTCATGCCGCGGTCACCAATCACA AAAAGTGGCAGTATAACTCCCCTCTGGTCCCGCGTAACGCTGAACT CGGGGACCGAAAAGGAAAAATTCACATCCCGTTTCCGCTGGCAAAT GTAACATGCATGGTGCCTAAAGCAAGGAACCCCACCGTGACGTACG GGAAAAACCAAGTCATCATGCTACTGTATCCTGACCACCCAACACT CCTGTCCTACCGGAGTATGGGAGAAGAACCAAACTATCAAGAAGA GTGGGTGACGCACAAGAAGGAGGTCGTGCTAACCGTGCCGACTGA AGGGCTCGAGGTTACGTGGGGCAACAACGAGCCGTATAAGTATTG GCCGCAGTTATCTGCAAACGGTACAGCCCACGGCCACCCGCATGAG ATAATCTTGTACTATTATGAGCTGTACCCTACTATGACTGTAGTAGT TGTGTCAGTGGCCTCGTTCATACTCCTGTCGATGGTGGGTATGGCAG TGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCATACGA ACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCT GCATCAGAACAGCTAAAGCGGCCACATACCAAGAGGCTGCGGTAT ACCTGTGGAACGAGCAGCAACCTTTGTTTTGGCTACAAGCCCTTATT CCGCTGGCAGCCCTGATTGTCCTATGCAACTGTCTGAGACTCTTACC ATGCTGTTGTAAAACGTTGGCTTTTTTAGCCGTAATGAGCATCGGTG CCCACACTGTGAGCGCGTACGAACACGTAACAGTGATCCCGAACAC GGTGGGAGTACCGTATAAGACTCTAGTCAACAGACCGGGCTACAGC CCCATGGTACTGGAGATGGAGCTACTGTCAGTCACTTTGGAGCCAA CGCTATCGCTTGATTACATCACGTGCGAATACAAAACCGTCATCCC GTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGCAAGGACAA AAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTACCCA TTTATGTGGGGCGGCGCCTACTGCTTCTGCGACGCTGAAAACACGC AATTGAGCGAAGCACATGTGGAGAAGTCCGAATCATGCAAAACAG AATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAA GCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTGCCTAT GCAAACGGCGACCATGCCGTCACAGTTAAGGACGCCAAATTCATTG TGGGGCCAATGTCTTCAGCCTGGACACCTTTTGACAACAAAATCGT GGTGTACAAAGGTGACGTTTACAACATGGACTACCCGCCCTTTGGC GCAGGAAGACCAGGACAATTTGGCGATATCCAAAGTCGCACGCCT GAGAGCAAAGACGTCTATGCTAACACACAACTGGTACTGCAGAGA CCGGCTGCGGGTACGGTACACGTGCCATACTCTCAGGCACCATCTG GCTTTAAGTATTGGTTAAAAGAACGAGGGGCGTCGCTACAGCACAC AGCACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGAT GAACTGCGCCGTAGGGAACATGCCCATCTCCATCGACATACCGGAT GCGGCCTTCACTAGGGTCGTCGACGCGCCCTCTTTAACGGACATGT CATGCGAGGTACCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGT CGCCATTATTAAATATGCAGTCAGCAAGAAAGGCAAGTGTGCGGTG CATTCGATGACCAACGCCGTCACTATCCGGGAAGCTGAGATAGAAG TTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACGGCCTTGGCC AGCGCCGAATTCCGCGTACAAGTCTGTTCTACACAAGTACACTGTG CAGCCGAGTGCCACCCTCCGAAGGACCACATAGTCAACTACCCGGC GTCACATACCACCCTCGGGGTCCAGGACATTTCCGCTACGGCGATG TCATGGGTGCAGAAGATCACGGGAGGTGTGGGACTGGTTGTCGCTG TTGCAGCACTGATTCTAATCGTGGTGCTATGCGTGTCGTTCAGCAGG CACTAACTTGACGACTAAGCATGAAGGTATATGTGTCCCCTAAGAG ACACACCGTATATAGCTAATAATCTGTAGATCAAAGGGCTATATAA CCCCTGAATAGTAACAAAATACAAAATCACTAAAAATTATAAAAA AAAAAAAAAAAAAACAGAAAAATATATAAATAGGTATACGTGTCC CCTAAGAGACACATTGTATGTAGGTGATAAGTATAGATCAAAGGGC CGAACAACCCCTGAATAGTAACAAAATATAAAAATTAATAAAAAT CATAAAATAGAAAAACCATAAACAGAAGTAGTTCAAAGGGCTATA AAAACCCCTGAATAGTAACAAAACATAAAACTAATAAAAATCAAA TGAATACCATAATTGGCAAACGGAAGAGATGTAGGTACTTAAGCTT CCTAAAAGCAGCCGAACTCACTTTGAGATGTAGGCATAGCATACCG AACTCTTCCACGATTCTCCGAACCCACAGGGACGTAGGAGATGTTA TTTTGTTTTTAATATTTC SEQ ID NO: 2 MEFIPTQTFYNRRYQPRPWTPRPTIQVIRPRPRPQRQAGQLAQLISAVN KLTMRAVPQQKPRKNRKNKKQKQKQQAPQNNTNQKKQPPKICKPAQ KKKKPGRRERMCMKIENDCIFEVKHEGKVTGYACLVGDKVMKPAHV KGTIDNADLAKLAFKRSSKYDLECAQIPVIIMKSDASKFTHEKPEGYYN WHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEG ARTALSVVTWNKDIVTKITPEGAEEWSLAIPVMCLLANTTFPCSQPPCI PCCYEKEPEETLRMLEDNVMRPGYYQLLQASLTCSPHRQRRSTKDNF NVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQI GIGTDDSHDWTKLRYMDNHIPADAGRAGLFVRTSAPCTITGTMGHFIL ARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKEL PCSTYVQSNAATAEEIEVHIMPPDTPDRTLLSQQSGNVKITVNSQTVRY KCNCGGSNEGLITTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRN AELGDRKGKIHIPFPLANVTCMVPKARNPTVTYGKNQVIMLLYPDHPT LLSYRSMGEEPNYQEEWVTHKKEVVLTVPTEGLEVTWGNNEPYKYW PQLSANGTAHGHPHEIILYYYELYPTMTVVVVSVASFILLSMVGMAVG MCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAATYQEAAVYLWN EQQPLFWLQALIPLAALIVLCNCLRLLPCCCKTLAFLAVMSIGAHTVSA YEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITC EYKTVIPSPYVKCCGTAECKDKNLPDYSCKVFTGVYPFMWGGAYCFC DAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITV TAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYP PFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPS GFKYWLKERGASLQHTAPFGCQIATNPVRAMNCAVGNMPISIDIPDAA FTRVVDAPSLTDMSCEVPACTHSSDFGGVAIIKYAVSICKGKCAVHSMT NAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAECHIPP KDHIVNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVV LCVSFSRH SEQ ID NO: 3 MDPVYVDIDADSAFLKALQRAYPMFEVEPRQVTPNDHANARAFSHLA IKLIEQEIDPDSTILDIGSAPARRMMSDRKYHCVCPMRSAEDPERLANY ARKLASAAGKVLDRNISGKIGDLQAVMAVPDTETPTFCLHTDVSCRQR ADVAIYQDVYAVHAPTSLYHQAIKGVRLAYWVGFDTTPFMYNAMAG AYPSYSTNWADEQVLKAKNIGLCSTDLTEGRRGKLSIMRGKKLEPCDR VLFSVGSTLYPESRKLLKSWHLPSVFHLKGKLSFTCRCDTVVSCEGYV VKRITMSPGLYGKTTGYAVTHHADGFLMCKTTDTVDGERVSFSVCTY VPATICDQMTGILATEVTPEDAQKLLVGLNQRIVVNGRTQRNTNTMK NYMIPVVAQAFSKWAKECRKDMEDEKLLGVRERTLTCCCLWAFKKQ KTHTVYKRPDTQSIQKVQAEFDSFVVPSLWSSGLSIPLRTRIKWLLSKV PKTDLTPYSGDAQEARDAEKEAEEEREAELTLEALPPLQAAQEDVQVE IDVEQLEDRAGAGIIETPRGAIKVTAQPTDHVVGEYLVLSPQTVLRSQK LSLIHALAEQVKTCTHSGRAGRYAVEAYDGRVLVPSGYAISPEDFQSL SESATMVYNEREFVNRKLHHIAMHGPALNIDEESYELVRAERTEHEY VYDVDQRRCCKKEEAAGLVLVGDLTNPPYHEFAYEGLKIRPACPYKIA VIGVFGVPGSGKSAIIKNLVTRQDLVTSGKKENCQEITTDVMRQRGLEI SARTVDSLLLNGCNRPVDVLYVDEAFACHSGTLLALIALVRPRQKVVL CGDPKQCGFFNMMQMKVNYNHNICTQVYHKSISRRCTLPVTAIVSSL HYEGKMRTTNEYNKPIVVDTTGSTKPDPGDLVLTCFRGWVKQLQIDY RGHEVMTAAASQGLTRKGVYAVRQKVNENPLYASTSEHVNVLLTRT EGKLVWKILSGDPWIKTLQNPPKGNFKATIKEWEVEHASIMAGICSHQ MTFDTFQNKANVCWAKSLVPILETAGIKLNDRQWSQIIQAFKEDKAYS PEVALNEICTRMYGVDLDSGLFSKPLVSVYYADNHWDNRPGGKMFGF NPEAASILERKYPFTKGKWNINKQICVTTRRIEDFNPTTNIIPANRRLPH SLVAEHRPVKGERMEWLVNKINGHHVLLVSGCSLALPTKRVTWVAPL GVRGADYTYNLELGLPATLGRYDLVVINIHTPFRIHHYQQCVDHAMK LQMLGGDSLRLLKPGGSLLIRAYGYADRTSERVICVLGRKFRSSRALK PPCVTSNTEMFFLFSNFDNGRRNFTTHVMNNQLNAAFVGQATRAGCA PSYRVKRMDIAKNDEECVVNAANPRGLPGDGVCKAVYKKWPESFKN SATPVGTAKTVMCGTYPVIHAVGPNFSNYSESEGDRELAAAYREVAK EVTRLGVNSVAIPLLSTGVYSGGKDRLTQSLNHLFTAMDSTDADVVIY CRDKEWEKKISEAIQMRTQVELLDEHISIDCDVVRVHPDSSLAGRKGY STTEGALYSYLEGTRFHQTAVDMAEIYTMWPKQTEANEQVCLYALGE SIESIRQKCPVDDADASSPPKTVPCLCRYAMTPERVTRLRMNHVTSIIV CSSFPLPKYKIEGVQKVKCSKVMLFDHNVPSRVSPREYRPSQESVQEA STTTSLTHSQFDLSVDGKILPVPSDLDADAPALEPALDDGAIHTLPSAT GNLAAVSDWVMSTVPVAPPRRRRGRNLTVTCDEREGNITPMASVRFF RAELCPVVQETAETRDTAMSLQAPPSTATELSHPPISFGAPSETFPITFG DFNEGEIESLSSELLTFGDFLPGEVDDLTDSDWSTCSDTDDELRLDRAG GYIFSSDTGPGHLQQKSVRQSVLPVNTLEEVHEEKCYPPKLDEAKEQL LLKKLQESASMANRSRYQSRKVENMKATIIQRLKRGCRLYLMSETPK VPTYRTTYPAPVYSPPINVRLSNPESAVAACNEFLARNYPTVSSYQITD EYDAYLDMVDGSESCLDRATFNPSKLRSYPKQHAYHAPSIRSAVPSPF QNTLQNVLAAATKRNCNVTQMRELPTLDSAVFNVECFKKFACNQEY WEEFAASPIRITTENLTTYVTKLKGPKAAALFAKTHNLLPLQEVPMDR FTVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRR LNAVLLPNVHTLFDMSAEDFDAIIAAHFKPGDTVLETDIASFDKSQDDS LALTALMLLEDLGVDHSLLDLIEAAFGEISSCHLPTGTRFKFGAMMKS GMFLTLFVNTLLNITIASRVLEDRLTKSACAAFIGDDNIIHGVVSDELM AARCATWMNMEVKIIDAVVSQKAPYFCGGFILHDIVTGTACRVADPL KRLFKLGKPLAAGDEQDEDRRRALADEVVRWQRTGLIDELEKAVYSR YEVQGISVVVMSMATFASSRSNFEKLRGPVVTLYGGPK SEQ ID NO: 4 ATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTAC TCTGCTTAGCAAGAGACTTGAGAACCCATCATGGATCCCGTGTACG TGGACATAGACGCCGACAGCGCCTTTTTAAAGGCCCTGCAGCGTGC GTACCCCATGTTTGAGGTGGAACCAAGGCAGGTCACACCGAATGAC CATGCCAATGCTAGAGCATTCTCGCATCTAGCTATAAAACTAATAG AGCAGGAAATTGATCCCGACTCAACCATCCTGGACATAGGCAGCGC GCCAGCAAGGAGGATGATGTCGGATAGGAAGTACCACTGCGTTTGC CCTATGCGCAGCGCAGAAGACCCTGAGAGACTCGCCAACTACGCG AGAAAACTAGCATCTGCCGCAGGAAAAGTCTTGGACAGAAACATC TCCGAAAAAATTGGAGATCTACAAGCAGTAATGGCTGTACCAGACG CAGAAACGCCCACATTCTGCTTGCACACTGACGTCTCATGTAGACA AAGGGCGGACGTCGCTATATACCAGGATGTCTACGCCGTGCATGCA CCAACATCGCTGTACCACCAGGCGATTAAAGGAGTCCGTGTAGCAT ACTGGATAGGGTTTGATACAACCCCGTTCATGTATAATGCCATGGC AGGTGCATACCCCTCGTACTCGACAAACTGGGCAGATGAGCAGGTG CTGAAGGCAAAGAACATAGGATTATGTTCAACAGACCTGACGGAA GGTAGACGAGGTAAATTGTCTATCATGAGAGGAAAAAAGATGAAG CCATGTGACCGCGTACTGTTCTCAGTCGGGTCAACGCTTTACCCGG AGAGCCGTAAGCTTCTTAAGAGTTGGCACTTACCTTCAGTGTTCCAT CTAAAAGGGAAGCTCAGCTTCACGTGCCGCTGTGATACAGTGGTTT CGTGTGAAGGCTATGTCGTTAAGAGAATAACGATTAGCCCGGGCCT CTACGGTAAAACCACAGGGTACGCAGTAACCCACCATGCAGACGG ATTCCTAATGTGCAAAACAACCGATACGGTAGATGGCGAGAGAGT GTCATTTTCGGTATGCACGTACGTACCCGCAACCATTTGTGATCAAA TGACAGGTATTCTTGCCACGGAGGTTACACCGGAGGATGCACAGAA GCTGCTGGTGGGACTGAACCAGAGGATAGTGGTCAATGGCAGAAC GCAGAGGAACACGAACACAATGAAGAATTACTTGCTTCCTGTAGTT GCCCAAGCCTTCAGTAAGTGGGCAAAGGAATGCCGGAAAGATATG GAAGATGAAAAACTTTTGGGCATCAGAGAAAGGACACTGACATGC TGCTGCCTTTGGGCGTTCAAGAAGCAGAAGACACACACGGTCTACA AGAGGCCTGACACTCAGTCAATTCAGAAAGTCCCAGCCGAATTTGA CAGCTTTGTGGTACCAAGTCTGTGGTCATCTGGACTGTCGATCCCGC TACGGACCAGAATCAAGTGGCTGCTAAGCAAAGTGCCAAAGACTG ATTTGATCCCTTACAGCGGTGACGCCAAAGAAGCCCGCGACGCTGA AAAAGAAGCAGAAGAAGAACGAGAAGCGGAGCTAACTCGCGAGG CACTACCACCACTACAGGCGGCACAGGACGACGTCCAGGTCGAAA TTGACGTGGAACAGCTCGAAGACAGAGCTGGGGCAGGAATAATTG AAACTCCAAGAGGAGCTATCAAAGTCACTGCCCAACCAACAGACC ACGTCGTGGGAGAGTACTTGGTACTTTCCCCGCAGACCGTGTTACG AAGCCAGAAGCTCAGCCTGATCCACGCATTGGCGGAACAAGTGAA GACATGCACACACAGCGGACGGGCAGGAAGGTACGCGGTCGAAGC ATATGACGGCAGAATCCTTGTGCCCTCAGGCTATGCAATATCACCT GAAGACTTCCAGAGCCTGAGCGAAAGTGCGACGATGGTGTACAAC GAAAGGGAGTTCGTAAATAGGAAATTACACCATATCGCGTTGCACG GACCAGCCCTGAACACTGACGAGGAGTCGTACGAGCTGGTAAGGG CAGAAAGGACAGAGCATGAGTACGTCTATGATGTGGACCAAAGAA GGTGCTGCAAGAAAGAGGAGGCAGCCGGGCTGGTACTGGTCGGCG ACTTGACCAACCCGCCCTACCATGAGTTCGCATATGAAGGGCTGAG AATCCGCCCCGCCTGCCCATACAAGACCGCAGTAATAGGGGTCTTT GGAGTGCCAGGATCCGGCAAATCAGCAATCATTAAGAACCTAGTTA CCAGGCAAGACCTAGTGACCAGTGGAAAGAAAGAAAACTGCCAAG AAATCTCCACCGACGTGATGCGACAGAGGAACCTGGAGATATCTGC ACGCACGGTCGACTCACTGCTCTTGAACGGATGCAATAGACCAGTC GACGTGTTGTACGTCGACGAAGCTTTTGCGTGCCATTCTGGCACGCT ACTTGCTCTGATAGCCTTGGTGAGACCGAGGCAGAAAGTCGTGCTA TGCGGTGATCCGAAACAGTGCGGCTTCTTCAATATGATGCAGATGA AAGTTAACTACAACCATAACATCTGCACCCAAGTGTACCATAAAAG TATTTCCAGGCGGTGTACACTGCCTGTGACTGCCATTGTGTCCTCGT TGCATTACGAAGGCAAAATGCGCACAACAAATGAGTACAACAAGC CAATTGTAGTGGATACTACAGGCTCGACAAAACCCGACCCCGGAGA CCTTGTGCTAACATGTTTCAGAGGGTGGGTTAAGCAACTGCAAATT GACTATCGTGGACACGAGGTCATGACAGCAGCTGCATCTCAGGGGC TAACCAGAAAAGGGGTCTATGCCGTCAGGCAAAAAGTTAATGAAA ACCCCCTTTACGCATCAACATCAGAGCACGTGAACGTGCTACTGAC GCGTACGGAAGGCAAACTAGTATGGAAGACACTTTCTGGAGACCC ATGGATAAAGACACTGCAGAACCCGCCGAAAGGAAATTTTAAAGC AACAATTAAGGAATGGGAAGTGGAACATGCTTCAATAATGGCGGG TATCTGTAACCACCAAGTGACCTTTGACACGTTCCAGAATAAAGCC AATGTCTGCTGGGCGAAGAGCTTAGTCCCCATCCTAGAAACAGCAG GGATAAAATTAAACGACAGGCAGTGGTCCCAGATAATCCAGGCTTT TAAAGAAGACAGAGCATACTCACCCGAGGTGGCCCTGAATGAGAT ATGCACGCGCATGTACGGGGTAGACCTGGACAGCGGACTGTTCTCT AAACCACTGGTGTCCGTGCATTATGCGGATAATCACTGGGACAACA GGCCGGGAGGGAAGATGTTCGGATTCAACCCCGAAGCGGCGTCCA TACTGGAGAGGAAATACCCGTTTACAAAAGGGAAGTGGAATACCA ACAAGCAAATCTGTGTGACTACTAGGAGGATTGAAGATTTTAACCC GAACACCAACATTATACCTGCCAACAGGAGATTACCGCATTCATTG GTGGCCGAACATCGCCCGGTAAAAGGGGAGAGGATGGAATGGTTG GTCAACAAAATAAATGGCCACCATGTGCTCCTGGTCAGCGGCTACA ACCTCGTTCTGCCCACTAAGAGAGTCACCTGGGTGGCGCCGCTGGG CATTCGGGGAGCTGACTACACATACAACCTAGAGTTAGGCCTACCA GCAACGCTCGGTAGATATGACCTAGTGATTATAAACATCCACACAC CCTTTCGCATACATCATTACCAACAGTGCGTGGATCACGCAATGAA GCTGCAGATGCTCGGAGGAGACTCCCTGAGACTGCTCAAGCCGGGT GGTTCATTACTGATCAGGGCATACGGCTACGCAGACAGAACAAGCG AACGAGTAGTCTGCGTATTGGGACGCAAGTTTCGATCATCCAGAGC GTTGAAACCGCCGTGCGTCACTAGCAACACCGAGATGTTTTTCTTGT TCAGCAACTTTGATAACGGCAGAAGGAACTTTACGACGCACGTAAT GAACAACCAGCTGAATGCTGCTTTTGTTGGTCAGGCCACCCGAGCA GGGTGCGCACCGTCGTACCGGGTTAAACGCATGGACATCGCAAAG AACGATGAAGAGTGTGTAGTCAACGCCGCCAACCCTCGTGGGCTAC CAGGCGATGGCGTCTGTAAAGCAGTATACAAAAAATGGCCGGAGT CCTTCAAGAACAGTGCAACACCAGTGGGAACCGCAAAGACAGTCA TGTGCGGTACATACCCGGTAATCCATGCAGTAGGACCTAATTTCTC AAATTACTCTGAGTCCGAAGGAGACCGGGAATTGGCAGCTGCTTAC CGAGAAGTCGCTAAGGAGGTGACTAGACTAGGAGTAAACAGCGTA GCTATACCGCTCCTTTCCACCGGTGTGTACTCTGGAGGGAAAGACA GGCTGACTCAGTCACTAAACCACCTTTTTACAGCATTAGACTCAACT GATGCAGATGTGGTTATCTACTGCCGCGACAAGGAGTGGGAGAAG AAAATAGCTGAGGCCATACAAATGAGGACCCAAGTGGAATTACTA GACGAACACATCTCTGTAGACTGCGATATCATCCGAGTGCACCCTG ACAGCAGTTTGGCAGGTAGAAAAGGGTACAGCACTACAGAAGGTT CACTGTACTCCTACTTGGAAGGGACACGGTTCCATCAGACGGCAGT GGACATGGCAGAAGTATACACCATGTGGCCAAAGCAGACGGAGGC TAATGAACAAGTTTGCTTGTACGCATTGGGGGAAAGTATAGAATCA ATCAGGCAAAAGTGCCCAGTGGATGACGCAGATGCATCGTCGCCCC CAAAAACCGTCCCGTGCCTCTGCCGTTATGCCATGACACCCGAACG AGTCACCAGGCTTCGTATGAACCATGTCACAAGCATAATAGTATGC TCATCATTCCCCCTTCCAAAGTATAAAATAGAAGGAGTGCAGAAAG TCAAGTGTTCTAAAGTGATGCTGTTCGACCATAACGTGCCATCACG CGTTAGTCCAAGGGAATATAAATCGCCTCAGGAGACCGCACAAGA AGTAAGTTCGACCACGTCACTGACGCACAGCCAATTCGACCTTAGC GTTGACGGTGAGGAACTGCCCGCTCCGTCTGACTTGGAAGCTGACG CTCCGATTCCGGAACCAACACCAGACGACAGAGCGGTACTTACTTT GCCTCCCACGATTGATAATTTTTCGGCTGTGTCAGACTGGGTAATGA ATACCGCGCCAGTCGCACCACCCAGAAGAAGACGTGGGAAAAACT TGAATGTCACCTGCGACGAGAGAGAAGGGAACGTACTTCCCATGGC TAGCGTTCGGTTCTTCAGAGCGGATCTGCACTCCATCGTACAGGAA ACGGCAGAGATACGCGATACGGCCGCGTCCCTCCAGGCGCCCCTGA GTGTCGCTACAGAACCGAATCAACTGCCGATCTCATTTGGAGCACC AAACGAGACTTTCCCCATAACGTTCGGGGATTTTGATGAAGGGGAG ATTGAAAGCTTGTCCTCTGAGTTACTGACCTTTGGGGACTTCTCGCC GGGCGAAGTGGATGACCTGACAGACAGCGACTGGTCCACGTGTTCA GACACGGACGACGAATTATGACTAGATAGGGCAGGTGGGTACATA TTCTCATCTGACACCGGCCCCGGCCACCTGCAACAGAGGTCTGTCC GTCAGACAGTACTGCCGGTAAATACCTTGGAGGAAGTTCAGGAGG AGAAATGTTACCCACCTAAGTTGGATGAAGTGAAAGAGCAGTTGTT ACTTAAGAAACTCCAGGAAAGTGCGTCCATGGCTAACAGAAGCAG GTACCAATCCCGCAAAGTAGAGAACATGAAAGCAACAATAGTCCA AAGGCTGAAGGGTGGTTGCAAACTTTATTTAATGTCGGAGACCCCG AAAGTTCCTACCTACCGAACTACATATCCGGCACCAGTGTACTCAC CCCCAATCAATATCCGACTGTCCAACCCCGAGTCTGCTGTGGCAGC GTGCAATGAGTTCCTAGCAAGGAACTATCCGACAGTTGCGTCGTAC CAAATCACCGATGAGTACGATGCATACCTAGACATGGTGGACGGGT CGGAAAGTTGCCTTGACCGGGCGACGTTCAACCCATCAAAGCTTAG AAGTTATCCAAAACAGCACTCCTACCATGCACCCACAATCAGAAGT GCCGTACCTTCCCCGTTCCAGAACACGCTGCAGAACGTACTGGCTG CTGCCACGAAAAGAAATTGCAACGTCACACAGATGAGAGAACTGC CTACTTTGGATTCAGCGGTATTTAATGTTGAGTGCTTTAAAAAATTT GCGTGCAATCAAGAATACTGGAAGGAATTTGCCGCCAGCCCTATTA GGATAACGACTGAGAACTTGACAACTTATGTCACAAAACTAAAAG GACCAAAAGCAGCAGCACTGTTTGCCAAGACACATAACCTGCTACC ACTGCAGGAGGTGCCGATGGACAGGTTTACTGTAGACATGAAAAG GGACGTGAAGGTGACTCCGGGGACGAAGCACACTGAGGAAAGACC TAAAGTGCAGGTCATACAGGCAGCCGAACCTTTGGCAACAGCATAT CTGTGTGGGATCCACAGAGAGTTGGTCAGAAGGCTGAATGCAGTCC TTCTACCTAATGTACACACGCTGTTTGACATGTCTGCCGAGGACTTT GACGCCATTATTGCCGCGCACTTCAAGCCGGGGGACGCCGTATTGG AAACCGATATAGCCTCCTTTGACAAGAGCCAAGACGACTCATTGGC GCTCACTGCTCTAATGTTGCTAGAGGATTTGGGGGTGGATCATCCC CTGTTGGACTTGATAGAGGCTGCCTTCGGGGAGATCTCCAGCTGCC ACCTACCGACGGGCACCCGTTTTAAGTTCGGCGCCATGATGAAGTC TGGTATGTTCCTAACCCTGTTCGTCAACACACTGCTAAACATCACCA TAGCCAGCCGAGTGCTGGAGGACCGCTTGACAAGGTCTGCGTGCGC GGCCTTCATCGGCGACGACAATATAATACATGGGGTTGTCTCTGAC GAACTGATGGCAGCAAGGTGTGCTACATGGATGAACATGGAAGTG AAGATCATAGATGCGGTCGTGTCTCAGAAAGCCCCGTACTTCTGCG GAGGGTTTATACTGTATGACACAGTAGCAGGCACGGCCTGCAGAGT GGCAGACCCGCTAAAGCGGCTGTTCAAGCTGGGCAAACCGCTGGC AGCGGGAGATGAACAAGACGACGACAGAAGACGTGCACTGGCTGA CGAAGTGGTTAGATGGCAACGAACAGGACTAACTGATGAGCTAGA AAAAGCGGTACACTCCAGGTATGAAGTGCAGGGCATATCTGTCGTG GTAATGTCTATGGCCACCTTTGCAAGCTCTAGATCTAACTTTGAGAA GCTCAGAGGACCCGTCGTAACCCTGTACGGTGGTCCTAAATAGGTA CGCACTACAGCTACCTATTTCGTCAGAAACCAATCGCAGCTACTTG CATACCTACCAGCTACAATGGAGTTCATCCCGACGCAAACTTTCTA TAACAGAAGGTACCAACCCCGACCCTGGGCCCCACGCCCTACAATT CAAGTAATTAGACCTAGACCACGTCCACAGAGGCAGGCTGGGCAA CTCGCCCAGCTGATCTCCGCAGTCAACAAATTGACCATGCGCGCGG TACCTCAACAGAAGCCTCGCAGAAATCGGAAAAACAAGAAGCAAA GGCAGAAGAAGCAGGCGCCGCAAAACGACCCAAAGCAAAAGAAG CAACCACCACAAAAGAAGCCGGCTCAAAAGAAGAAGAAACCAGGC CGTAGGGAGAGAATGTGCATGAAAATTGAAAATGATTGCATCTTCG AAGTCAAGCATGAAGGCAAAGTGATGGGCTACGCATGCCTGGTGG GGGATAAAGTAATGAAACCAGCACATGTGAAGGGAACTATCGACA ATGCCGATCTGGCTAAACTGGCCTTTAAGCGGTCGTCTAAATACGA TCTTGAATGTGCACAGATACCGGTGCACATGAAGTCTGATGCCTCG AAGTTTACCCACGAGAAACCCGAGGGGTACTATAACTGGCATCACG GAGCAGTGCAGTATTCAGGAGGCCGGTTCACTATCCCGACGGGTGC AGGCAAGCCGGGAGACAGCGGCAGACCGATCTTCGACAACAAAGG ACGGGTGGTGGCCATCGTCCTAGGAGGGGCCAACGAAGGTGCCCG CACGGCCCTCTCCGTGGTGACGTGGAACAAAGACATCGTCACAAAA ATTACCCCTGAGGGAGCCGAAGAGTGGAGCCTCGCCCTCCCGGTCT TGTGCCTGTTGGCAAACACTACATTCCCCTGCTCTCAGCCGCCTTGC ACACCCTGCTGCTACGAAAAGGAACCGGAAAGCACCTTGCGCATGC TTGAGGACAACGTGATGAGACCCGGATACTACCAGCTACTAAAAGC ATCGCTGACTTGCTCTCCCCACCGCCAAAGACGCAGTACTAAGGAC AATTTTAATGTCTATAAAGCCACAAGACCATATCTAGCTCATTGTCC TGACTGCGGAGAAGGGCATTCGTGCCACAGCCCTATCGCATTGGAG CGCATCAGAAATGAAGCAACGGACGGAACGCTGAAAATCCAGGTC TCTTTGCAGATCGGGATAAAGACAGATGACAGCCACGATTGGACCA AGCTGCGCTATATGGATAGCCATACGCCAGCGGACGCGGAGCGAG CCGGATTGCTTGTAAGGACTTCAGCACCGTGCACGATCACCGGGAC CATGGGACACTTTATTCTCGCCCGATGCCCGAAAGGAGAGACGCTG ACAGTGGGATTTACGGACAGCAGAAAGATCAGCCACACATGCACA CACCCGTTCCATCATGAACCACCTGTGATAGGTAGGGAGAGGTTCC ACTCTCGACCACAACATGGTAAAGAGTTACCTTGCAGCACGTACGT GCAGAGCACCGCTGCCACTGCTGAGGAGATAGAGGTGCATATGCCC CCAGATACTCCTGACCGCACGCTGATGACGCAGCAGTCTGGCAACG TGAAGATCACAGTTAATGGGCAGACGGTGCGGTACAAGTGCAACT GCGGTGGCTCAAACGAGGGACTGACAACCACAGACAAAGTGATCA ATAACTGCAAAATTGATCAGTGCCATGCTGCAGTCACTAATCACAA GAATTGGCAATACAACTCCCCTTTAGTCCCGCGCAACGCTGAACTC GGGGACCGTAAAGGAAAGATCCACATCCCATTCCCATTGGCAAACG TGACTTGCAGAGTGCCAAAAGCAAGAAACCCTACAGTAACTTACGG AAAAAACCAAGTCACCATGCTGCTGTATCCTGACCATCCGACACTC TTGTCTTACCGTAACATGGGACAGGAACCAAATTACCACGAGGAGT GGGTGACACACAAGAAGGAGGTTACCTTGACCGTGCCTACTGAGG GTCTGGAGGTCACTTGGGGCAACAACGAACCATACAAGTACTGGCC GCAGATGTCTACGAACGGTACTGCTCATGGTCACCCACATGAGATA ATCTTGTACTATTATGAGCTGTACCCCACTATGACTGTAGTCATTGT GTCGGTGGCCTCGTTCGTGCTTCTGTCGATGGTGGGCACAGCAGTG GGAATGTGTGTGTGCGCACGGCGCAGATGCATTACACCATATGAAT TAACACCAGGAGCCACTGTTCCCTTCCTGCTCAGCCTGCTATGCTGC GTCAGAACGACCAAGGCGGCCACATATTACGAGGCTGCGGCATATC TATGGAACGAACAGCAGCCCCTGTTCTGGTTGCAGGCTCTTATCCC GCTGGCCGCCTTGATCGTCCTGTGCAACTGTCTGAAACTCTTGCCAT GCTGCTGTAAGACCCTGGCTTTTTTAGCCGTAATGAGCATCGGTGCC CACACTGTGAGCGCGTACGAACACGTAACAGTGATCCCGAACACG GTGGGAGTACCGTATAAGACTCTTGTCAACAGACCGGGTTACAGCC CCATGGTGTTGGAGATGGAGCTACAATCAGTCACCTTGGAACCAAC ACTGTCACTTGACTACATCACGTGCGAGTACAAAACTGTCATCCCC TCCCCGTACGTGAAGTGCTGTGGTACAGCAGAGTGCAAGGACAAG AGCCTACCAGACTACAGCTGCAAGGTCTTTACTGGAGTCTACCCAT TTATGTGGGGCGGCGCCTACTGCTTTTGCGACGCCGAAAATACGCA ATTGAGCGAGGCACATGTAGAGAAATCTGAATCTTGCAAAACAGA GTTTGCATCGGCCTACAGAGCCCACACCGCATCGGCGTCGGCGAAG CTCCGCGTCCTTTACCAAGGAAACAACATTACCGTAGCTGCCTACG CTAACGGTGACCATGCCGTCACAGTAAAGGACGCCAAGTTTGTCGT GGGCCCAATGTCCTCCGCCTGGACACCTTTTGACAACAAAATCGTG GTGTACAAAGGCGACGTCTACAACATGGACTACCCACCTTTTGGCG CAGGAAGACCAGGACAATTTGGTGACATTCAAAGTCGTACACCGG AAAGTAAAGACGTTTATGCCAACACTCAGTTGGTACTACAGAGGCC AGCAGCAGGCACGGTACATGTACCATACTCTCAGGCACCATCTGGC TTCAAGTATTGGCTGAAGGAACGAGGAGCATCGCTACAGCACACG GCACCGTTCGGTTGCCAGATTGCGACAAACCCGGTAAGAGCTGTAA ATTGCGCTGTGGGGAACATACCAATTTCCATCGACATACCGGATGC GGCCTTTACTAGGGTTGTCGATGCACCCTCTGTAACGGACATGTCAT GCGAAGTACCAGCCTGCACTCACTCCTCCGACTTTGGGGGCGTCGC CATCATCAAATACACAGCTAGCAAGAAAGGTAAATGTGCAGTACAT TCGATGACCAACGCCGTTACCATTCGAGAAGCCGACGTAGAAGTAG AGGGGAACTCCCAGCTGCAAATATCCTTCTCAACAGCCCTGGCAAG CGCCGAGTTTCGCGTGCAAGTGTGCTCCACACAAGTACACTGCGCA GCCGCATGCCACCCTCCAAAGGACCACATAGTCAATTACCCAGCAT CACACACCACCCTTGGGGTCCAGGATATATCCACAACGGCAATGTC TTGGGTGCAGAAGATTACGGGAGGAGTAGGATTAATTGTTGCTGTT GCTGCCTTAATTTTAATTGTGGTGCTATGCGTGTCGTTTAGCAGGCA CTAAACCGATGATAAGGCACGAAATAACTAAATAGCAAAAGTAGA AAGTACATAACCAGGTATATGTGCCCCTTAAGAGGCACAATATATA TAGCTAAGCACTATTAGATCAAAGGGCTATACAACCCCTGAATAGT AACAAAACACAAAAACCAATAAAAATCATAAAAAGAAAAATCTCA TAAACAGGTATAAGTGTCCCCTAAGAGACACATTGTATGTAGGTAG TAAGTATAGATCAAAGGGCTATATTAACCCCTGAATAGTAACAAAA CACAAAAACAATAAAAACTACAAAATAGAAAATCTATAAACAAAA GTAGTTCAAAGGGCTACAAAACCCCTGAATAGTAACAAAACATAA AATGTAATAAAAATTAAGTGTGTACCCAAAAGAGGTACAGTAAGA ATCAGTGAATATCACAATTGGCAACGAGAAGAGACGTAGGTATTTA AGCTTCCTAAAAGCAGCCGAACTCACTTTGAGACGTAGGCATAGCA TACCGAACTCTTCCACTATTCTCCGAACCCACAGGGACGTAGGAGA TGTTATTTTGTTTTTAATATTTC SEQ ID NO: 5 MEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVN KLTMRAVPQQKPRRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQ KKKKPGRRERMCMKIENDCIFEVKHEGKVMGYACLVGDKVMKPAHV KGTIDNADLAKLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYN WHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEG ARTALSVVTWNKDIVTKITPEGAEEWSLALPVLCLLANTTFPCSQPPCT PCCYEKEPESTLRMLEDNVMRPGYYQLLKASLTCSPHRQRRSTKDNF NVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGI KTDDSHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILA RCPKGETLTVGFTDSRKISHTCTHPFHHEPPVIGRERFHSRPQHGKELPC STYVQSTAATAEEIEVIEVIPPDTPDRTLMTQQSGNVKITVNGQTVRYKC NCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAEL GDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLS YRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQ MSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTAVGMC VCARRRCITPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYLWNEQ QPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYE HVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEY KTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDA ENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAA YANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPF GAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGF KYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTR VVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTNA VTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPK DHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLC VSFSRH SEQ ID NO: 6 MDPVYVDIDADSAFLKALQRAYPMFEVEPRQVTPNDHANARAFSHLA IKLIEQEIDPDSTILDIGSAPARRMMSDRKYHCVCPMRSAEDPERLANY ARKLASAAGKVLDRNISEKIGDLQAVMAVPDAETPTFCLHTDVSCRQR ADVAIYQDVYAVHAPTSLYHQAIKGVRVAYWIGFDTTPFMYNAMAG AYPSYSTNWADEQVLKAKNIGLCSTDLTEGRRGKLSIMRGKKMKPCD RVLFSVGSTLYPESRKLLKSWHLPSVFHLKGKLSFTCRCDTVVSCEGY VVKRITISPGLYGKTTGYAVTHHADGFLMCKTTDTVDGERVSFSVCTY VPATICDQMTGILATEVTPEDAQKLLVGLNQRIVVNGRTQRNTNTMK NYLLPVVAQAFSKWAKECRKDMEDEKLLGIRERTLTCCCLWAFKKQ KTHTVYKRPDTQSIQKVPAEFDSFVVPSLWSSGLSIPLRTRIKWLLSKV PKTDLIPYSGDAKEARDAEKEAEEEREAELTREALPPLQAAQDDVQVE IDVEQLEDRAGAGIIETPRGAIKVTAQPTDHVVGEYLVLSPQTVLRSQK LSLIHALAEQVKTCTHSGRAGRYAVEAYDGRILVPSGYAISPEDFQSLS ESATMVYNEREFVNRKLHHIALHGPALNTDEESYELVRAERTEHEYV YDVDQRRCCKKEEAAGLVLVGDLTNPPYHEFAYEGLRIRPACPYKTA VIGVFGVPGSGKSAIIKNLVTRQDLVTSGKKENCQEISTDVMRQRNLEI SARTVDSLLLNGCNRPVDVLYVDEAFACHSGTLLALIALVRPRQKVVL CGDPKQCGFFNMMQMKVNYNHNICTQVYHKSISRRCTLPVTAIVSSL HYEGKMRTTNEYNKPIVVDTTGSTKPDPGDLVLTCFRGWVKQLQIDY RGHEVMTAAASQGLTRKGVYAVRQKVNENPLYASTSEHVNVLLTRT EGKLVWKTLSGDPWIKTLQNPPKGNFKATIKEWEVEHASIMAGICNH QVTFDTFQNKANVCWAKSLVPILETAGIKLNDRQWSQIIQAFKEDRAY SPEVALNEICTRMYGVDLDSGLFSKPLVSVHYADNHWDNRPGGKMFG FNPEAASILERKYPFTKGKWNINKQICVTTRRIEDFNPNTNIIPANRRLP HSLVAEHRPVKGERMEWLVNKINGHHVLLVSGYNLVLPTKRVTWVA PLGIRGADYTYNLELGLPATLGRYDLVIINIHTPFRIHHYQQCVDHAMK LQMLGGDSLRLLKPGGSLLIRAYGYADRTSERVVCVLGRKFRSSRALK PPCVTSNTEMFFLFSNFDNGRRNFTTHVMNNQLNAAFVGQATRAGCA PSYRVKRMDIAKNDEECVVNAANPRGLPGDGVCKAVYKKWPESFKN SATPVGTAKTVMCGTYPVIHAVGPNFSNYSESEGDRELAAAYREVAK EVTRLGVNSVAIPLLSTGVYSGGKDRLTQSLNHLFTALDSTDADVVIY CRDKEWEKKIAEAIQMRTQVELLDEHISVDCDIIRVHPDSSLAGRKGYS TTEGSLYSYLEGTRFHQTAVDMAEVYTMWPKQTEANEQVCLYALGE SIESIRQKCPVDDADASSPPKTVPCLCRYAMTPERVTRLRMNHVTSIIV CSSFPLPKYKIEGVQKVKCSKVMLFDHNVPSRVSPREYKSPQETAQEV SSTTSLTHSQFDLSVDGEELPAPSDLEADAPIPEPTPDDRAVLTLPPTID NFSAVSDWVMNTAPVAPPRRRRGKNLNVTCDEREGNVLPMASVRFF RADLHSIVQETAEIRDTAASLQAPLSVATEPNQLPISFGAPNETFPITFGD FDEGEIESLSSELLTFGDFSPGEVDDLTDSDWSTCSDTDDELLDRAGGY IFSSDTGPGHLQQRSVRQTVLPVNTLEEVQEEKCYPPKLDEVKEQLLLK KLQESASMANRSRYQSRKVENMKATIVQRLKGGCKLYLMSETPKVPT YRTTYPAPVYSPPINIRLSNPESAVAACNEFLARNYPTVASYQITDEYD AYLDMVDGSESCLDRATFNPSKLRSYPKQHSYHAPTIRSAVPSPFQNT LQNVLAAATKRNCNVTQMRELPTLDSAVFNVECFKKFACNQEYWKE FAASPIRITTENLTTYVTKLKGPKAAALFAKTHNLLPLQEVPMDRFTVD MKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCGIHRELVRRLNA VLLPNVHTLFDMSAEDFDAIIAAHFKPGDAVLETDIASFDKSQDDSLAL TALMLLEDLGVDHPLLDLIEAAFGEISSCHLPTGTRFKFGAMMKSGMF LTLFVNTLLNITIASRVLEDRLTRSACAAFIGDDNIIHGVVSDELMAARC ATWMNMEVKIIDAVVSQKAPYFCGGFILYDTVAGTACRVADPLKRLF KLGKPLAAGDEQDDDRRRALADEVVRWQRTGLTDELEKAVHSRYEV QGISVVVMSMATFASSRSNFEKLRGPVVTLYGGPK SEQ ID NO: 7 ATGGCGGGCAAGTGACACTTGTTCCGCCGGACGTCTCTAAGCTCTT CCTCTGCATTGCAAGAGTTTACCACTCAGTATGTCGAAAGTCTTTGT AGATATCGAGGCCGAGAGCCCGTTTTTAAAATCACTACAGAGAGCG TTTCCAGCATTTGAAGTGGAAGCACAGCAGGTTACACCAAATGACC ATGCTAACGCCAGAGCATTCTCGCATCTGGCTACTAAATTGATAGA GCAAGAGACCGAAAAAGACACACTCATCCTGGATATCGGCAGTGC GCCTGCCAGGAGAATGATGTCTGAGCACACGTACCATTGTGTGTGC CCAATGCGCAGCGCTGAGGACCCAGAGCGTCTGCTGTATTATGCCA GGAAGTTAGCCAAGGCATCAGGTGAAGTCGTTGACAGAAATATTGC AGCGAAGATAGACGACCTGCAGTCAGTGATGGCGACCCCGGACAA TGAGTCACGGACATTTTGCCTTCACACAGATCAGACATGCAGGACT CCAGCTGAGGTGGCAGTCTATCAGGATGTCTATGCAGTTCACGCAC CGACTTCTTTGTACTTCCAGGCAATGAAAGGAGTACGCACAGCGTA CTGGATTGGGTTCGACACTACCCCATTCATGTTCGATACAATGGCC GGGGCTTATCCAACATATGCAACCAACTGGGCCGACGAACAGGTGT TGAAAGCCAGGAACATAGGACTGTGCTCCGCGGCACTGACTGAGG GACACCTTGGCAAGCTATCAATAATGAGGAAGAAGAGAATGAAAC CGAGTGACCAGATAATGTTCTCGGTAGGTTCCACATTGTATACGGA AAGCCGACGTCTGCTAAAGAGTTGGCATCTGCCGTCAGTGTTTCAT CTGAAAGGACGACAATCATATACGTGCCGGTGCGATACAATAGTGT CATGTGAGGGCTACGTTGTTAAGAAGATAACGATGAGCCCAGGGGT ATTCGGAAAAACGTCAGGGTACGCCGTCACACATCACGCAGAAGG ATTTCTGGTATGTAAAACCACTGACACTATCGCAGGAGAACGAGTT TCGTTCCCCGTTTGCACGTATGTGCCGTCGACGATCTGTGACCAAAT GACAGGCATTCTGGCGACTGAGGTCACGCCCGAGGATGCGCAAAA ATTGCTTGTCGGATTAAACCAGAGGATCGTCGTGAATGGGAGGACC CAGAGAAACACTAATACAATGAAGAACTACCTGCTCCCAGTGGTGT CACAAGCATTTAGTAAGTGGGCGAAAGAGTATCGGCTTGACCAAG ACGATGAGAAAAACATGGGTATGCGCGAACGCACCCTGACTTGCTG CTGCCTGTGGGCCTTTAAGACCCATAAGAATCACACCATGTATAAG AAGCCAGACACACAGACGATCGTCAGTGTCCCGTCGGAGTTCAACT CCTTCGTGATCCCTAGCCTGTGGTCAGCGGGGTTGTCGATCGGAATT AGACACAGGATCAGGCTTCTTTTGCAGTCAAGACGTGCCGAGCCGC TCGTTCCATTTATGGATGCTAGTGAAGCCAGAGCAGCAGAGAAGGA GGCTGCAGAAGCTAAGGAGGCAGAAGAGACACTGGCAGCCCTACC ACCTCTGATCCCGACCGCTCCGCTGCTCGATGACATCCCTGAAGTTG ATGTAGAGGAGCTGGAGTTTCGAGCAGGAGCCGGTGTTGTCGAAAC GCCTCGGAACGCCCTCAAAGTTACACCACAAGACAGAGACACTATG GTAGGTAGCTATCTCGTTCTGTCTCCCCAGACAGTCCTGAAAAGTGT TAAACTGCAGGTACTGCACTCCTTGGCAGAGCAAGTAAGAATCATT ACCCACAAAGGCCGTGCAGGGCGCTACCAAGTGGATGCCTATGAC GGCCGTGTTTTAATCCCGACAGGCGCGGCTATCCCGGTACCCGATT TCCAGGCTCTAAGTGAGAGTGCGACTATGGTGTACAACGAACGTGA GTTTATCAATCGCAAATTGTACCACATAGCCGTACATGGGGCAGCC CTGAACACCGATGAGGAGGGTTACGAGAAAGTGCGGGCCGAAAGG ACAGATGCCGAGTACGTTTTTGACGTGGATCGCAAACAGTGCGTTA AGAGGGAGGATGCCGAGGGTCTCGTGATGATCGGAGACCTGGTCA ACCCACCATTCCACGAATTTGCGTATGAAGGACTGAAACGAAGACC AGCGGCTCCATACAAAACAACTGTAGTCGGAGTCTTCGGCGTGCCT GGATCTGGAAAATCGGGTATAATCAAAAGCCTGGTCACACGTGCGG ACTTGGTCACCAGTGGAAAGAGAGAGAACTGTCAGGAGATCATGC ACGACGTCAAGAGATACAGAGACCTGGACATCACTGCAAAAACGG TGGATTCCGTACTGTTGAATGGCGTTAAGCAGACTGTCGACGTCTT GTACGTCGATGAGGCTTTCGCTTGCCATGCAGGGACATTGCTAGCG CTTATCGCCACGGTGCGACCACGTAAGAAGGTTGTGCTTTGCGGCG ACCCGAAACAGTGCGGTTTCTTCAACTTGATGCAACTACAGGTTAA CTTTAACCACAACATCTGCACCGAGGTACACCATAAGAGCATTTCC AGAAGATGCACGCTACCAGTCACCGCCATTGTCTCAACACTACACT ATGAGGGCAAAATGCGCACCACTAACCCATACAATAAACCCGTTGT CATTGACACCACAGGCCAGACTAAACCAAACCGCGAGGACATTGT GTTAACATGTTTCCGCGGTTGGGTTAAGCAGCTGCAACTTGACTATC GTGGACACGAAGTCATGACAGCCGCTGCATCCCAGGGGTTGACCCG AAAGGGCGTGTACGCCGTCCGAATGAAGGTCAACGAAAACCCACT GTACGCACAATCATCGGAGCATGTTAATGTTCTACTGACACGCACA GAGGGCAGACTAGTGTGGAAGACACTGTCAGGAGATCCCTGGATC AAAACTTTGAGCAACATCCCAAAAGGGAATTTCACAGCAACTTTGG AAGATTGGCAACAAGAGCACGACGCCATTATGAGGGCAATAACAC AAGAAGCAGCCCCTTTGGACGTGTTCCAAAATAAGGCTAAGGTGTG TTGGGCCAAGTGCTTGGTACCCGTTTTGGAAACCGCGGGGATCAAG TTGTCAGCCGCCGATTGGAGCTCAATCATTTTGGCTTTCAAAGAAG ACAGAGCTTATTCACCAGAGGTTGCACTGAATGAGATTTGCACTAA AATCTACGGGGCGGATTTGGACAGCGGCCTGTTTTCGGCTCCACGC GTGTCGCTACACTATACTACAAATCATTGGGATAACTCGCCTGGAG GAAGGATGTACGGGTTTTCCGTCGAGGCCGCCAACCGCCTAGAACA ACGGCACCCGTTCTACAGGGGACGGTGGGCTTCTGGGCAGGTGTTG GTCGCAGAACGAAGAACTCAGCCGATTGACATCACTTGCAACCTAA TCCCCTTCAACCGGAGACTCCCACACGCGCTGGTCACGGAATATCA TCCAGTTAAGGGAGAAAGAGTGGAGTGGCTTGTGAATAAGATCCC AGGCTATCACTTGCTACTGGTTAGCGAGTATAACCTCATACTGCCTA GAAGGAAGGTAACGTGGATTGCCCCGCCGACTGTGACAGGAGCCG ATTTAACCCACGACTTGGATTTAGGACTACCGCCTAATGCTGGCAG GTATGACCTAGTCTTCGTCAACATGCATACACCGTATAGGCTCCATC ACTACCAACAATGCGTCGATCACGCCATGAAATTACAGATGCTGGG CGGCGACGCACTCTACCTGTTGAAACCCGGGGGAAGCCTCCTTTTG AGAGCCTACGGTTATGCCGATAGAACGAGCGAGGCTGTGGTGACG GCTCTCGCTCGTCGGTTCTCGTCCTTCAGAGCGGTCAGACCTCCATG TGTGACTAGTAACACCGAGGTGTTCTTACTGTTCACGAACTTTGACA ACGGTAGAAGAACAGTAACCCTGCATCCTACAAATGGTAAATTATC ATCAATTTATGCAGGTACAGTGCTGCAGGCGGCCGGCTGCGCTCCC GCTTATACTGTCAAAAGGGCAGACATCGCGACCGCCATTGAGGATG CGGTGGTCAATGCAGCTAACCACCGTGGACAAGTGGGCGACGGAG TCTGCAGGGCAGTAGCACGGAAATGGCCTCAAGCCTTCCGCAACGC AGCGACACCTGTCGGAACCGCAAAAACCGTCAAGTGCGACGAGAC TTACATCATCCACGCGGTGGGTCCAAATTTTAACAATACATCTGAG GCTGAAGGGGATCGTGACTTGGCGGCGGCATACAGGGCCGTAGCA GCGGAGATTAACCGACTGTCTATAAGTAGTGTGGCGATTCCACTGC TTTCCACAGGTATATTCAGTGCAGGAAAAGATAGAGTGCATCAGTC GCTTTCGCACCTTCTAGCGGCAATGGACACCACTGAAGCACGGGTT ACTATCTACTGCCGCGATAAAACGTGGGAGCAAAAGATCAAAACA GTTCTGCAGAATCGCTGCGCCACTGAACTGGTGTCTGATGTGCTAC AGCTTGAAGTCAATTTGACCAGAGTCCATCCGGACAGCAGCCTGGT GGGACGTCCAGGGTATAGCACCACTGATGGGACCTTATATTCCTAT ATGGAAGGTACTAAGTTCCACCAAGCGGCTCTTGACATGGCTGAGA TCACGACCTTGTGGCCGAGAGTTCAGGATGCAAATGAACACATCTG CATGTATGCACTGGGCGAGACGATGGACAACATCCGCTCTAGATGC CCAGTTGAGGATAGTGACTCATCGACTCCACCGAAGACAGTCCCAT GTCTCTGTCGGTACGCTATGACACCAGAGAGAGTCACAAGACTACG AATGCATCACACAAAAGATTTTGTGGTCTGCTCGTCTTTCCAGCTAC CGAAGTACCGCATAGCTGGTGTGCAGCGAGTAAAGTGCGAAAAAG TGATGCTTTTTGATGCAACTCCACCGGCCTCTGTTAGTCCTGTGCAA TACCTGACGAGTCACAGTGAAACTACTGTAAGCTTGAGCTCGTTCT CAATTACATCTGACAGCAGCTCCCTAAGCACCTTCCCGGATCTGGA GTCACTAGAAGAACTGGGCAATGATCCACAGTCCATGCGGATGGAC GAGTCTGACAACCGGCAACCCATATCAACGGTAGAACCGGTTGTTC GACCCGTACCACCTCCGCGTCCTAAACGTGCCAGGCGACTAGCGGC TGCACGTATGCAGGTCCAGGCGGAAGTGCACCACCCACCCGTCGTC CAAAGGACGAAACCGGTCCCAGCACCGCGCACCAGTTTGCGTCCCG TCCCCGCGCCCAGAAGGTGTATGCCAAGACCAGCAGTAGAGCTGCC CTGGCCGCAGGAGGCCGTCGACATAGAGTTCGGGGCGCCGACCGA AGAGGAGAGTGAAATCACATTCGGAGACTTTTCTGCTTCGGAGTGG GAGACCATCAGCAACTCATCCTGACTAGGCCGAGCGGGGGCTTATA TCTTCTCATCAGACGTCGGTCCAGGGCATCTGCAACAGAAATCAGT GAGACAGCACGATCTAGAGGTGCCGATTATGGATCGCGTAGTCGAG GAGAAAGTCTACCCGCCGAAACTAGATGAGGCAAAAGAGAAACAG CTGCTCCTAAAACTGCAGATGCATGCCACAGACGCCAACCGGAGCC GGTACCAATCAAGGAAAGTTGAGAACATAAAAGCAACGATCATTG ACCGGCTGAAACAAGGCAGCGCATCCTACATCTCGGCTGAGGCCAA TAAAGCAATCACATACCATGTCAAATATGCTAAGCCTCGGTACTCT GTGCCGGTGATGCAAAGACTTAGCAGTGCAACCACCGCAGTTGCCG CTTGCAATGAATTCCTGGCCCGGAACTACCCTACAGTGGCGTCATA TCAGATCACCGATGAGTACGACGCTTATTTAGATATGGTGGACGGG TCAGAAAGCTGCCTAGACAGAGCAAACTTCTGCCCGGCGAAGTTGC GCTGCTATCCAAAACACCATGCATACCATGTACCCCAGATTAGAAG TGCTGTTCCTTCGCCATTCCAAAACACGTTGCAGAATGTATTAGCGG CCGCCACTAAGCGTAATTGCAACGTCACCCAGATGCGTGAACTACC AACCCTGGATTCAGCCGTGTACAACGTGGAATGTTTCCGCAAGTAC GCCTGTAACAACGAATATTGGGAAGAGTTTGCTGCTAAACCTATCA GAATTACAACAGAGAATTTGACCACTTATGTGACCAAATTAAAAGG TGGAAAGGCAGCCGCCCTGTTTGCAAAGACGCATAACTTAGTTCCA CTGCAGGAGGTTCCAATGGATAGATTCGTCATGGACATGAAGCGCG ATGTGAAGGTTACACCAGGGACGAAGCACACAGAGGAACGACCAA AGGTCCAAGTGATTCAAGCTGCCGAGCCTCTGGCTACCGCCTACCT GTGTGGAATTCACAGAGAACTGGTTCGCCGGCTCAATGCTGTGTTG CTACCTAACATCCATACCCTGTTTGACATGTCTGCTGAAGATTTTGA TGCCATCATATCAGAGCACTTCAAGCCAGGGGACCATGTCTTGGAA ACGGACATCGCTTCTTTTGACAAAAGCCAGGACGATTCACTGGCAC TAACGGGGCTAATGATACTGGAAGACTTGGGCGTCGATAACCAGTT ATTGGATCTTATCGAGGCTGCATTTGGTCAGATTACCAGCTGCCACC TGCCTACAGGGACTAGGTTTAAATTTGGGGCTATGATGAAGTCAGG CATGTTTCTTACATTGTTCATTAACACCGTTTTGAACATTACCATTG CCAGTAGAGTGCTGGAAGCCAGATTAACTAACTCAGCCTGTGCCGC ATTTATCGGCGACGACAACGTGGTTCACGGAGTCGTCTCCGATAAA CTGATGGCAGATAGATGTGCCACTTGGGTTAACATGGAGGTTAAAA TAATAGATGCAGTCATGTGTGCAAAGCCACCGTATTTCTGTGGAGG CTTTTTGGTCTATGATCATGTCACAAGGACGTCATGTCGAATAGCG GATCCATTAAAAAGGTTATTCAAATTGGGCAAACCCCTGCCGGCAG ACGACTGCCAAGATGAAGACCGCCGTAGGGCATTGCACGACGAGG TTAAAAAATGGTTTAGATCAGGCTTGGGTTCGGAGATTGAGGTCGC CCTCGCCACCAGATACGAGGTGGAAGGGGGTCACAACCTACTGCTG GCTATGTCCACCTTTGCACACAGCATGAAGAATTTTTCTGCATTGAG GGGACCCGTCATACACTTGTACGGCGGTCCTAAATAGGTGCTCTAC ACGACACCTATACCACCATGGATTTCCTACCAACACAAGTGTTTTAT GGCAGGCGATGGAGACCACGAATGCCGCCACGCCCTTGGAGGCCA CGCCCACCTACAATTCAAAGACCAGATCAACAGGCCCGACAAATG CAGCAGCTGATTGCTGCGGTCAGTACGCTTGCCCTTAGGCAAAACG CTGCTGCCCCTCAGCGTGGAAGAAAGAAGCAACCACGTAGAAAGA AACCAAAACCACAACCCGAGAAACCTAAGAAGCAAGAGCAGAAAC CGAAGCAAAAGAAGACCCCTAAGAAGAAGCCCGGGAGAAGGGAG CGCATGTGCATGAAGATTGAGCACGATTGCATCTTTGAGGTCAAGC ACGAAGGAAAGGTCACAGGCTATGCTTGCCTTGTCGGTGACAAAGT AATGAAGCCGGCACACGTCCCTGGAGTGATAGACAACATCGATCTC GCACGTCTATCGTATAAGAAATCTAGTAAGTATGACCTGGAATGTG CACAGATACCGGTGGCTATGAAGTCAGATGCATCGAAATACACCCA TGAAAAACCCGAAGGTCACTATAACTGGCACTATGGGGCCGTCCAG TACACAGGAGGCAGATTCACGGTGCCCACAGGAGTGGGTAAGCCT GGCGACAGCGGCCGGCCCATCTTTGATAACAAGGGCCGCGTTGTCG CAATAGTGCTAGGAGGAGCTAATGAAGGTGCCAGAACCGCGCTCTC TGTCGTGACGTGGAATAAAGACATGGTCACCAAGATCACACCGGA GGGCACAGAGGAGTGGGCAGCTCCGACAGTGACAGCTATGTGCCTT CTGGCGAATGTATCCTTCCCATGTTTCCAACCGAGCTGCTCTCCATG CTGTTATGAAAAGGGGCCTGAGCCGACGCTGAGAATGCTGGAAGA GAACGTAAACTCAGAGGGATACTATGAATTGTTGCATGCTGCCGTG TACTGCAAAAACAGCTCGAGGTCGAAGAGGAGCACTGCAAATCAC TTTAACGCATACAAATTGACTCGTCCATACGTAGCCTACTGTGCAG ACTGTGGCATGGGCCATTCTTGCCACAGTCCCGCCATGATTGAAAA TGTGCAGGCGGATGCTACAGATGGCACGTTAAAGATTCAATTCGCT TCTCAAATTGGCCTAACGAAAGCGGACACGCATGATCATACAAAGA TTAGATATGCAGAAGGGCATGATATAGCGGAGGCTGCCAGGTCAA CTCTTAAGGTCCATAGCAGCAGTGAGTGCGCGGTGACAGGAACGAT GGGACACTTTATCTTGGCCAAATGTCCACCAGGCGAAGTCATTAGT GTCTCATTTGTTGATTCAAAAAATGAACAGCGGACCTGTCGGATAG CCTACCACCATGAACAGAGGCTAATAGGGCGTGAAAGATTCACGGT ACGACCACATCATGGGATTGAGCTACCCTGCACCACTTACCAGCTA ACGACCGCCGAAACCTCTGAAGAGATAGACATGCACATGCCGCCG GACATTCCGGACAGAACTATCCTTTCCCAGCAATCAGGAAATGTCA AGATAACGGTCAACGGGCGGACCGTCAAGTATAGCTGCTCATGTGG TTCTAAACCATCAGGCACAACAACTACAGACAAGACTATCAATAGC TGCACCGTTGACAAATGCCAGGCATATGTCACGAGCCATACGAAAT GGCAATTTAATTCACCTTTCGTTCCGCGCGCGGAGCAAGCGGAGCG TAAGGGCAAGGTGCACATTCCCTTTCCACTTATCAACACCACCTGC CGAGTACCATTGGCTCCCGAAGCCCTAGTCAGGAGCGGTAAACGAG AAGCTACGCTCTCACTGCACCCGATACATCCCACATTGCTAAGTTA CAGAACACTGGGACGCGAGCCAGTTTTTGATGAACAGTGGATCACC ACCCAGACGGAGGTAACAATCCCAGTACCAGTGGAGGGAGTGGAG TATCGGTGGGGCAACCACAAACCACAACGCTTGTGGTCACAGCTAA CAACTGATGGCAGAGCACATGGCTGGCCCCATGAAATTATCGAGTA CTACTACGGGCTGCATCCTACGACAACCATTGTCGTGGTGGTTGCT GTCTCAGTAGTGGTGCTCTTGTCAGTTGCTGCATCTGTTTATATGTG TGTAGTGGCACGCAACAAGTGCCTGACACCATATGCACTCACCCCG GGGGCCGTTGTCCCTGTTACTATCGGGGTATTATGCTGTGCACCGA AAGCGCACGCAGCTAGCTTTGCTGAAGGCATGGCCTACCTGTGGGA TAATAACCAGTCAATGTTCTGGATGGAGTTGACTGGACCTTTGGCT CTTCTTATCCTGACTACATGTTGCGCCCGATCACTACTTTCCTGCTG CAAGGGATCTTTTTTAGTCGCAGTGAGCGTCGGGAGTGCCGTTGCC AGTGCTTACGAGCACACGGCAGTCATTCCAAATCAAGTGGGATTCC CGTATAAGGCTCATGTTGCACGTGAAGGATACAGCCCGCTGACACT GCAGATGCAGGTGGTTGAAACCAGTCTAGAGCCAACGCTCAACCTG GAGTATATCACTTGCGACTACAAAACAAAGGTTCCATCCCCGTACG TAAAGTGCTGTGGCACAGCTGAATGCCGCACGCAGGACAAGCCCG AATACAAGTGTGCGGTGTTCACGGGCGTGTACCCTTTTATGTGGGG AGGTGCATACTGTTTTTGTGACTCGGAAAACACACAGATGAGTGAG GCCTACGTGGAGCGCGCTGACGTGTGCAAACACGACTACGCAGCCG CCTACCGCGCCCACACTGCTTCCCTCAGAGCCAAAATCAAGGTAAC ATATGGCACCGTAAATCAGACTGTCGAGGCGTATGTGAACGGTGAC CATGCCGTAACGATTGCCGGGACGAAGTTTATCTTTGGTCCGGTGT CAACGGCCTGGACACCGTTCGATACTAAAATCGTGGTCTATAAAGG GGAGGTATACAACCAGGACTTTCCCCCTTATGGTGCCGGGCAGCCT GGAAGATTCGGAGACATCCAGAGTAGGACGTTGGACAGTAAGGAC CTATATGCAAACACGGGCCTTAAGCTGGCAAGACCAGCAGCCGGC AACATCCACGTCCCCTATACCCAGACACCATCTGGTTTTAAAACAT GGCAAAAAGACAGGGACTCACCGTTAAACGCTAAGGCACCCTTTG GATGCACAATCCAGACAAATCCGGTCAGAGCGATGAATTGCGCTGT CGGCAACATACCCGTTTCGATGGATATCGCCGACAGCGCATTCACT AGACTGACTGATGCGCCTATAATATCAGAGCTGCTGTGCACTGTAT CTACATGCACGCATTCTTCAGACTTCGGTGGAGTCGCTGTACTTTCT TACAAGGTGGAAAAAGCAGGCAGGTGCGACGTCCATTCGCACTCG AATGTCGCGGTACTCCAGGAAGTTTCCATCGAGGCAGAAGGTCGAT CAGTGATCCACTTTTCGACCGCATCAGCCGCCCCTTCCTTCATAGTA TCCGTCTGCAGCTCGCGTGCCACGTGCACAGCTAAATGTGAACCAC CGAAAGACCATGTGGTCACTTACCCGGCAAATCACAACGGGATAAC TTTGCCGGACTTATCCAGCACTGCAATGACTTGGGCGCAACATCTT GCCGGTGGAGTCGGGCTATTGATAGCACTGGCAGTGCTAATTCTAG TAATAGTTACTTGCATAACTTTGAGAAGGTGAATCATAAGTACCAT GTATAATGCGGGCTGGCATATATGTAACCATTATTATATATTTTAAC CATATTAACATTCGAATATCAAATTAGGTGCCGTGCATGATGCGGA CCGATTTATTATTCACATATGTAACCTTTATCATATTACATCATATA TTCAATTTTAAAATTTCTATACGCGTCTCTAATGGCGCATATAATAA CCACCTACAATTTTCTTCATTTTCTTTATTTGTGCCACTATAGGGCAC TTACTAACCATAGAAGTAATTCATTTTGTTTTTAATATTTC SEQ ID NO: 8 MDFLPTQVFYGRRWRPRMPPRPWRPRPPTIQRPDQQARQMQQLIAAV STLALRQNAAAPQRGRKKQPRRKKPKPQPEKPKKQEQKPKQKKTPKK KPGRRERMCMKIEHDCIFEVKHEGKVTGYACLVGDKVMKPAHVPGVI DNIDLARLSYKKSSKYDLECAQIPVAMKSDASKYTHEKPEGHYNWHY GAVQYTGGRFTVPTGVGKPGDSGRPIFDNKGRVVAIVLGGANEGART ALSVVTWNKDMVTKITPEGTEEWAAPTVTAMCLLANVSFPCFQPSCSP CCYEKGPEPTLRMLEENVNSEGYYELLHAAVYCKNSSRSKRSTANHF NAYKLTRPYVAYCADCGMGHSCHSPAMIENVQADATDGTLKIQFASQ IGLTKADTHDHTKIRYAEGHDIAEAARSTLKVHSSSECAVTGTMGHFIL AKCPPGEVISVSFVDSKNEQRTCRIAYHHEQRLIGRERFTVRPHHGIELP CTTYQLTTAETSEEIDMHNIPPDIPDRTILSQQSGNVKITVNGRTVKYSC SCGSKPSGTTITDKTINSCTVDKCQAYVTSHTKWQFNSPFVPRAEQAE RKGKVHIPFPLINTTCRVPLAPEALVRSGKREATLSLHPIHPTLLSYRTL GREPVFDEQWITTQTEVTIPVPVEGVEYRWGNHKPQRLWSQLTTDGR AHGWPHEIIEYYYGLHPTTTIVVVVAVSVVVLLSVAASVYMCVVARN KCLTPYALTPGAVVPVTIGVLCCAPKAHAASFAEGMAYLWDNNQSMF WMELTGPLALLILTTCCARSLLSCCKGSFLVAVSVGSAVASAYEHTAV IPNQVGFPYKAHVAREGYSPLTLQMQVVETSLEPTLNLEYITCDYKTK VPSPYVKCCGTAECRTQDKPEYKCAVFTGVYPFMWGGAYCFCDSENT QMSEAYVERADVCKHDYAAAYRAHTASLRAKIKVTYGTVNQTVEAY VNGDHAVTIAGTKFIFGPVSTAWTPFDTKIVVYKGEVYNQDFPPYGAG QPGRFGDIQSRTLDSKDLYANTGLKLARPAAGNIHVPYTQTPSGFKTW QKDRDSPLNAKAPFGCTIQTNPVRAMNCAVGNIPVSMDIADSAFTRLT DAPIISELLCTVSTCTHSSDFGGVAVLSYKVEKAGRCDVHSHSNVAVL QEVSIEAEGRSVIRESTASAAPSFIVSVCSSRATCTAKCEPPKDHVVTYP ANHNGITLPDLSSTAMTWAQHLAGGVGLLIALAVLILVIVTCITLRR SEQ ID NO: 9 MSKVFVDIEAESPFLKSLQRAFPAFEVEAQQVTPNDHANARAFSHLAT KLIEQETEICDTLILDIGSAPARRIVIMSEHTYHCVCPMRSAEDPERLLYY ARKLAKASGEVVDRNIAAKIDDLQSVMATPDNESRTFCLHTDQTCRTP AEVAVYQDVYAVHAPTSLYFQAMKGVRTAYWIGFDTTPFMFDTMAG AYPTYATNWADEQVLKARNIGLCSAALTEGHLGKLSIMRKKRMKPSD QIMDFSVGSTLYTESRRLLKSWHLPSVFHLKGRQSYTCRCDTIVSCEGY VVKKITMSPGVFGKTSGYAVTHHAEGFLVCKTTDTIAGERVSFPVCTY VPSTICDQMTGILATEVTPEDAQKLIVGLNQRIVVNGRTQRNTNTMKN YLLPVVSQAFSKWAKEYRLDQDDEKNMGMRERTLTCCCLWAFKTHK NHTMYKKPDTQTIVSVPSEFNSFVIPSLWSAGLSIGIRHRIRLLLQSRRA EPLVPFMDASEARAAEKEAAEAKEAEETLAALPPLIPTAPLLDDIPEVD VEELEFRAGAGVVETPRNALKVTPQDRDTMVGSYLVLSPQTVLKSVK LQVLHSLAEQVRIITHKGRAGRYQVDAYDGRVLIPTGAAIPVPDFQALS ESATMVYNEREFINRKLYHIAVHGAALNTDEEGYEKVRAERTDAEYV FDVDRKQCVKREDAEGLVMIGDLVNPPFHEFAYEGLKRRPAAPYKTT VVGVFGVPGSGKSGIIKSLVTRADLVTSGKRENCQEIMHDVKRYRDLD ITAKTVDSVLLNGVKQTVDVLYVDEAFACHAGTLLALIATVRPRKKV VLCGDPKQCGFFNLMQLQVNFNHNICTEVHHKSISRRCTLPVTAIVSTL HYEGKMRTTNPYNKPVVIDTTGQTKPNREDIVLTCFRGWVKQLQLDY RGHEVMTAAASQGLTRKGVYAVRMKVNENPLYAQSSEHVNVLLTRT EGRLVWKTLSGDPWIKTLSNIPKGNFTATLEDWQQEHDAIMRAITQEA APLDVFQNICAKVCWAKCLVPVLETAGIKLSAADWSSIILAFKEDRAYS PEVALNEICTKIYGADLDSGLFSAPRVSLHYTTNHWDNSPGGRMYGFS VEAANRLEQRHPFYRGRWASGQVLVAERRTQPIDITCNLIPFNRRLPH ALVTEYHPVKGERVEWLVNKIPGYHLLLVSEYNLILPRRKVTWIAPPT VTGADLTHDLDLGLPPNAGRYDLVFVNMHTPYRLHHYQQCVDHAMK LQMLGGDALYLLKPGGSLLLRAYGYADRTSEAVVTALARRFSSFRAV RPPCVTSNTEVFLLFTNFDNGRRTVTLHPTNGKLSSIYAGTVLQAAGC APAYTVKRADIATAIEDAVVNAANHRGQVGDGVCRAVARKWPQAFR NAATPVGTAKTVKCDETYIIHAVGPNFNNTSEAEGDRDLAAAYRAVA AEINRLSISSVAIPLLSTGIFSAGKDRVHQSLSHLLAAMDTTEARVTIYC RDKTWEQKIKTVLQNRCATELVSDVLQLEVNLTRVHPDSSLVGRPGY STTDGTLYSYMEGTKFHQAALDMAEITTLWPRVQDANEHICMYALGE TMDNIRSRCPVEDSDSSTPPKTVPCLCRYAMTPERVTRLRMHHTKDFV VCSSFQLPKYRIAGVQRVKCEKVMLFDATPPASVSPVQYLTSHSETTV SLSSFSITSDSSSLSTFPDLESLEELGNDPQSMRMDESDNRQPISTVEPVV RPVPPPRPKRARRLAAARMQVQAEVHHPPVVQRTKPVPAPRTSLRPVP APRRCMPRPAVELPWPQEAVDIEFGAPTEEESEITFGDFSASEWETISNS SLGRAGAYIFSSDVGPGHLQQKSVRQHDLEVPIMDRVVEEKVYPPKLD EAKEKQLLLKLQMHATDANRSRYQSRKVENIKATHDRLKQGSASYIS AEANKAITYHVKYAKPRYSVPVMQRLSSATTAVAACNEFLARNYPTV ASYQITDEYDAYLDMVDGSESCLDRANFCPAKLRCYPKHHAYHVPQI RSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTLDSAVYNVECFRK YACNNEYWEEFAAKPIRITTENLTTYVTKLKGGKAAALFAKTHNLVPL QEVPMDRFVMDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLC GIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIISEHFKPGDHVLETDIAS FDKSQDDSLALTGLMILEDLGVDNQLLDLIEAAFGQITSCHLPTGTRFK FGAMMKSGMFLTLFINTVLNITIASRVLEARLTNSACAAFIGDDNVVH GVVSDKLMADRCATWVNMEVKIIDAVMCAKPPYFCGGFLVYDHVTR TSCRIADPLKRLFKLGKPLPADDCQDEDRRRALHDEVKKWFRSGLGSE IEVALATRYEVEGGHNLLLAMSTFAHSMKNFSALRGPVIHLYGGPK SEQ ID NO: 10 TGGCGGACGTGTGACATCACCGTTCGCCTTTCTTGGATCCCTTGTTA CTCCACGTAGTGAGAGATAAACAACCCAGAATGAAGGTCACTGTA GATGTTGAGGCTGATAGCCCATTTTTGAAGGCCCTTCAGAAAGCAT TCCCGGCTTTTGAGGTTGAATCACAGCAGGTCACACCCAATGACCA TGCTAATGCCAGAGCTTTTTCGCATCTGGCCACAAAGTTAATTGAA CAAGAGGTTCCAACCAACATCACCATCCTGGATGTGGGCAGCGCGC CCGCAAGGAGGTTGATGTCTGATCACAGCTACCACTGCATTTGTCC CATGAAGAGTGCGGAAGACCCAGAGAGATTAGCGAACTATGCCCG GAAACTGGCAAAAGCAGCAGGGGAAGTGCTAGACAAAAATGTGTC CGGTAAGATCACGGACCTGCAGGACGTCATGGCAACCCCCGATCTG GAATCTCCAACGTTCTGTCTCCACACCGACGAGACGTGTCGCACTA GAGCTGAAGTAGCTGTGTACCAGGACGTGTACGCGGTGCACGCACC GACCTCGCTTTATCATCAAGCGATGAAAGGGGTCAGGACAGTATAC TGGATAGGATTTGACACCACCCCATTCATGTTCGAGGTTTTGGCTGG CGCATACCCAACGTATTCCACGAATTGGGCGGACGAGCAGGTCCTG CAGGCACGTAACATCGGCCTATGTGCGACCAGTCTCAGCGAAGGAC ATCGAGGAAAACTCTCTATTATGAGGAAGAAACGCCTAAGGCCAA GCGACAGGGTCATGTTCTCGGTTGGGTCAACGCTATATATAGAAAG TAGACGCCTTCTTAAGAGTTGGCATCTTCCCTCCGTGTTCCACCTGA AAGGCAAGAATAGCTTTACTTGCAGGTGCGACACAATAGTTTCATG CGAGGGCTACGTTGTTAAAAAGATCACAATGAGCCCAGGGACGTA CGGGAAGACGGTCGGATACGCCGTTACGCATCATGCAGAAGGTTTC CTAATGTGCAAGGTGACGGACACTGTGCGCGGGGAGAGAGTATCA TTCCCGGTATGCACTTATGTGCCTGCAACCATTTGCGATCAGATGAC AGGAATCCTGGCTACCGACGTCACACCCGAAGATGCGCAGAAACTC CTGGTGGGGTTGAACCAACGTATAGTTGTGAACGGCAGAACCCAAA GAAATACCAACACAATGAAAAACTACCTATTGCCGGTAGTGGCCCA AGCTTTCAGCAAGTGGGCACGAGAAGCAAAAGCAGACATGGAGGA TGAGAAACCTCTGGGAACCAGAGAAAGAACCCTGACGTGCTGTTGC CTGTGGGCATTCAAGAGCCACAAGACACACACCATGTACAAAAGG CCGGATACCCAAACTATAGTCAAGGTGCCATCTACTTTTGACTCGTT TGTGATTCCAAGCCTGTGGTCATCCAGCCTATCCATAGGCCTACGG CAAAGAATAAAACTGCTATTAGGCCCAAAACTCTCGCGGGACCTCC CGTACTCTGGAGACCGAAACGAAGCGCGAGAAGCAGAGAAGGAAG CCGAAGAGACCAAGGAAGCAGAATTGACACGGGAAGCACTGCCAC CATTGGTGGGAAGCAACTGCGCTGATGACGTCGATCGGGTAGATGT GGAAGAGCTGACGTACCGCGCCGGAGCAGGGGTAGTGGAGACACC CAGGAATGCGCTCAGAGTGACACCACAAGAGCGCGATCAGCTGAT CGGCGCGTACCTGATCTTGTCTCCGCAAGCAGTACTGAAGAGTGAA AAACTCACACCGATACATCCACTGGCTGAGCAAGTGACAATCATGA CGCACTCTGGAAGATCTGGCAGATACCCGGTTGACCGCTACGACGG ACGGGTGTTGGTCCCGACGGGCGCAGCGATCCCCGTCAGCGAATTT CAAGCACTCAGTGAGAGTGCTACTATGGTCTACAACGAACGTGAGT TCATCAATCGCAAGTTGCACCATATAGCACTCTACGGCCCCGCCCT AAACACTGAGGAGGAGAACTACGAAAAAGTAAGGGCAGAGAGAG CCGAAGCTGAATACGTGTTTGACGTTGACAAGAGGATGTGCGTGAA GAGAGAAGAAGCATCAGGCCTTGTACTGGTTGGTGACTTAATCAAC CCACCCTTCCATGAGTTCGCGTATGAAGGACTGAAGATACGGCCCG CAACGCCTTTCCAAACCACGGTCATTGGCGTCTTCGGGGTACCTGG TTCGGGCAAGTCAGCCATAATCAAGAGTGTGGTGACTACGAGGGAC CTGGTCGCCAGTGGGAAGAAGGAGAACTGCCAGGAGATAGTTAAT GATGTCAAAAAGCAGAGAGGACTGGATGTAACAGCCAGGACTGTT GACTCTATCCTGTTGAACGGGTGCAGAAGAGGAGTAGAGAACCTAT ACGTGGACGAAGCATTTGCCTGTCATTCAGGTACACTGTTGGCTCTT ATTGCCATGGTGAAACCAACTGGCAAGGTGATCCTATGCGGAGACC CAAAACAGTGTGGATTTTTTAACCTGATGCAACTGAAGGTGAACTT CAACCACGATATCTGCACACAAGTGCTCCACAAAAGCATATCCAGA AGATGCACCCTCCCGATCACGGCTATCGTCTCGACCCTACACTATC AGGGTAAGATGAGAACCACGAACTTATGTAGTGCACCCATCCAGAT AGACACAACAGGTACCACTAAACCAGCCAAAGGAGACATCGTGTT AACATGCTTCCGCGGTTGGGTAAAGCAACTGCAGATAGACTACCGT GGACACGAAGTCATGACAGCAGCTGCATCACAAGGACTGACTAGA AAAGGGGTATACGCCGTTAGGCAGAAAGTGAATGAAAACCCGCTT TATGCCCCTTCATCAGAACATGTCAACGTATTGCTGACTAGAACTG AAAACCGGCTGGTGTGGAAAACACTGTCGGGAGACCCGTGGATAA AGGTGTTAACCAATATCCCCAAGGGCGATTTCAGCGCTACGTTGGA GGAGTGGCAAGAGGAGCATGACAACATCATGAACGCCCTTCGCGA GAGGTCGACAGCAGTAGACCCGTTCCAGAACAAAGCCAAAGTCTG CTGGGCAAAGTGCCTCGTGCAGGTCCTAGAAACGGCTGGGATACGC ATGACGGCAGAGGAGTGGGATACAGTGTTGGCTTTCCGCGAAGAC AGGGCGTACTCACCCGAAGTGGCTCTGAACGAGATCTGTACCAAGT ATTACGGCGTTGACTTAGACAGCGGATTGTTCTCCGCCCAATCGGT CTCATTGTACTATGAAAACAACCACTGGGACAATAGACCGGGCGGC CGGATGTATGGATTCAACCGCGAAGTCGCCCGTAAGTTTGAGCAAC GCTACCCATTCCTGAGAGGCAAGATGGACTCGGGGCTACAAGTTAA TGTTCCAGAGAGAAAAGTACAGCCATTCAATGCGGAATGTAATATA TTACCATCAAACAGGCGACTTCCGCACGCCCTCGTCACCAGCTACC AGCAGTGCCAGGGCGAGAGGGTAGAATGGCTTTTGAAGAAGCTTC CCGGATACCATTTATTGCTGGTAAGCGAGTACAATCTGGCGCTGCC CCATAAAAGAGTCTTTTGGATTGCACCACCCCATGTGTCTGGTGCA GATCGTATTTATGATCTTGACCTAGGATTACCCCTGAATGCAGGCC GTTACGACTTGGTATTTGTGAACATACACACTGAGTACAGGACGCA CCACTACCAACAGTGCGTCGACCACTCTATGAAGTTACAGATGTTG GGCGGTGACTCCTTACATCTATTGAAACCAGGCGGCTCACTGCTTA TCCGTGCTTACGGGTACGCCGACAGAGTCAGCGAAATGGTGGTCAC TGCATTAGCTAGGAAGITTTCCGCCTTCAGAGTCTTGAGACCAGCA TGTGTAACAAGTAACACTGAAGTCTTTCTGTTGTTCACCAATTTTGA TAACGGCAGAAGGGCTGTGACTCTTCACCAGGCCAATCAGAGGCTC AGCTCCATGTTTGCATGCAACGGGCTACACACAGCCGGATGCGCAC CCTCATACCGTGTGCGTAGGACCGACATTTCCGGGCACGCTGAAGA GGCGGTTGTTAATGCCGCCAACGCGAAGGGCACAGTCGGCGATGG GGTTTGCAGAGCGGTGGCGAGAAAATGGCCAGACTCCTTCAAAGGT GCCGCGACTCCCGTGGGTACGGCTAAGTTGGTACAGGCCAACGGTA TGAATGTCATCCACGCGGTAGGCCCGAATTTCTCCACGGTGACCGA GGCAGAGGGCGACAGAGAGTTGGCCGCCGCATACCGTGCCGTGGC GGGTATTATCAATGCTAGTAACATTAAGAGTGTAGCCATCCCTCTG TTGTCGACGGGAGTGTTCTCCGGAGGTAAAGATAGAGTCATGCAGT CACTAAATCACCTGTTTACCGCAATGGACACCACGGACGCTGACGT AGTCATCTATTGCCGCGACAAAGCCTGGGAGAAGAAAATCCAGGA GGCTATCGATCGCCGCACCGCCGTGGAATTGGTATCTGAAGACATC TCACTCGAGTCTGACTTGATACGGGTACACCCAGATAGTTGCTTGG TAGGCAGAAAAGGTTACAGCATAACAGATGGGAAGCTGCATTCAT ACCTGGAAGGTACCCGCTTTCATCAGACTGCGGTGGACATGGCTGA GATATCTACCTTGTGGCCGAAACTTCAGGACGCAAACGAACAAATA TGCTTGTATGCATTGGGTGAGAGTATGGACAGCATCAGAACGAAAT GCCCTGTTGAGGACGCCGATTCGTCCACGCCTCCGAAAACAGTTCC GTGTCTGTGTAGGTACGCTATGACTGCTGAGAGAGTGGCAAGACTT CGGATGAACAACACTAAGGCCATAATTGTGTGCTCCTCCTTCCCTTT ACCGAAGTACAGGATTGAAGGCGTCCAGAAGGTCAAGTGCGACCG AGTGCTGATTTTTGACCAGACGGTGCCATCTCTGGTTAGTCCAAGG AAGTACATACCAGCCGCCGCCTCTACGCACGCAGATACCGTGAGCT TGGATTCTACAGTATCCACAGGATCCGCGTGGTCATTCCCATCTGA GGCCACGTATGAGACCATGGAAGTAGTAGCAGAGGTGCACCACTC GGAACCACCAGTCCCGCCACCGCGCAGGCGTCGTGCGCAGGTGAC GATGCACCACCAGGAGCTGTTGGAAGTCTCTGACATGCACACCCCG ATTGCGGCAAGGGTCGAGATCCCCGTGTACGATACCGCTGTTGTAG TGGAGAGAGTGGCAATTCCTTGCACAAGCGAGTATGCAAAACCCAT ACCAGCACCACGGGCAGCAAGGGTCGTACCCGTGCCGGCACCACG CATTCAGCGAGCGTCGACGTACAGAGTCTCTCCTACACCCACGCCT CGCGTTCTGAGAGCCTCGGTATGCAGTGTGACCACTAGCGCTGGGG TAGAGTTCCCTTGGGCGCCTGAAGATCTGGAGGTACTCACCGAGCC TGTGCACTGCAAAATGCGCGAGCCGGTTGAGTTACCGTGGGAGCCT GAGGACGTTGATATCCAGTTCGGAGATTTTGAAACATCCGACAAAA TCCAATTCGGCGATATTGATTTTGACCAATTCTGACTAGGCAGAGC GGGGGCGTACATCTTCTCGTCTGATACCGGACCAGGGCACTTACAA CAGAAGTCAGTACGGCAACACGCACTACCGTGCGAAATGCTATACG TCCACGAGGAAGAACGGACGTACCCCCCCGCACTGGATGAGGCCA GGGAGAAACTGCTGCAGGCAAAAATGCAGATGGCACCTACGGAAG CAAACAAGAGCAGGTACCAATCAAGGAAGGTTGAAAACATGAAGG CAGTGATCATAGATAGGCTGAAGGATGGAGCAAGAACCTACCTGA CAGAACAGTCAGAGAAGATTCCAACCTATGTTAGTAAGTACCCGCG GCCAGTTTACTCGCCGTCGGTAGAGGATAGCTTGCAGAATCCCGAG GTCGCTGTGGCGGCCTGCAATGCTTTCCTGGAAGCCAATTACCCGA CAGTGGCTAGTTACCAGATCACGGACGAGTATGATGCCTACTTGGA TATGGTTGATGGGTCAGAGAGTTGTTTAGACCGGGCAACCTTCTGC CCGGCAAAATTACGCTGCTACCCAAAGCATCATGCTTACCACCAAC CGCAGGTTAGGAGCGCGGTCCCATCACCATTTCAAAACACCCTGCA GAATGTGCTAGCAGCAGCCACGAAGAGAAACTGCAATGTTACACA GATGAGAGAGCTACCCACTCTAGACTCAGCCGTGCTTAACGTGGAA TGCTTCAAAAAATTCGCATGCAACGGAGAATACTGGCAGGAATTCA AAGACAACCCAATAAGAATAACTACAGAGAACATAACAACTTATG TTACTAGGCTTAAGGGCCCTAAAGCAGCGGCGCTGTTTGCAAAGAC TCACAATCTAGTCCCGCTGCAGGAGGTGCCCATGGACCGGTTTGTG GTAGATATGAAGAGAGACGTGAAAGTTACCCCTGGCACCAAACAT ACCGAGGAACGCCCAAAGGTGCAAGTCATCCAGGCCGCCGAACCT TTAGCTACAGCTTATTTATGTGGCATTCACAGGGAGTTAGTCCGCCG CCTGAAGGCCGTCCTGGCCCCGAACATACATACATTGTTCGATATG TCGGCAGAAGATTTTGATGCCATCATAGCTGCACATTTCCAACCAG GCGACGCAGTTTTGGAAACGGACATAGCCTCCTTTGACAAGAGCCA AGATGACTCTCTGGCGTTGACGGCACTGATGCTGTTGGAAGACCTC GGGGTTGACCAAGAACTACTAGACTTGATAGAGGCAGCGTTCGGG GAAATTACCAGCGTCCACCTGCCAACAGGTACGCGGTTCAAGTTTG GCGCCATGATGAAGTCCGGAATGTTCCTGACACTGTTTGTAAATAC CCTGTTAAACATTGTCATAGCATGCCGTGTACTGCGTGAGAAGCTG ACAAACTCCGTCTGCGCCGCGTTTATCGGGGATGACAACATAGTGC ACGGGGTAAGATCCGACCCGTTGATGGCTGAAAGGTGCGCCAGCTG GGTTAATATGGAGGTAAAGATAATTGACGCTACCATGTGCGAGAAA CCACCATATTTCTGCGGCGGGTTTATATTGTATGACAAAGTCACCG GATCGGCGTGCCGAGTGGCCGACCCTCTGAAAAGGTTATTTAAACT AGGTAAACCTTTACCCGCCGGAGACACCCAAGATGAAGATCGTAG GCGTGCATTGAAGGATGAGACGGATAGGTGGGCACGAGTAGGGCT GAAGTCTGAACTGGAAATAGCACTAAGTTCTCGGTATGAGGTGAAC GGGACCGGCAACATAGTGCGAGCAATGGCCACACTGGCCAAGAGC CTGAAGAATTTTAAAAAGCTGCGTGGACCCATCGTACACCTCTACG GCGGTCCTAAATAGATGCAGAGACACACCTTCATCTAATACAGCTC ACAACAGTAAACATGAATTACATACCAACCCAGACTTTTTACGGAC GCCGTTGGCGGCCTCGCCCGGCGTTCCGTCCATGGCAGGTGCCGAT GCAGCCGACACCTACTATGGTTACACCCATGCTGCAAGCACCAGAC CTACAGGCCCAACAGATGCAACAACTGATCAGCGCTGTCTCTGCAT TAACCACCAAACAGAATGTAAAAGCACCAAAAGGGCAACGGAAGA AGAAACAGCAGAAACCAAAGGAAAAGAAGGAAAACCAGAAGAAA AAGCCGACGCAAAAGAAGAAGCAGCAGCAGAAACCAAAACCACA GGCTAAGAAGAAGAAACCAGGGAGAAGAGAAAGAATGTGCATGA AGATCGAGAATGACTGCATATTCGAGGTCAAACTGGATGGCAAGGT TACCGGTTATGCGTGCCTAGTCGGAGACAAGGTCATGAAGCCGGCT CACGTTAAAGGCACAATTGATAACCCAGACCTTGCGAAGCTGACTT ACAAGAAATCCAGTAAGTATGACCTCGAATGCGCCCAGATACCAGT GCACATGAAGTCCGACGCCTCCAAGTACACACATGAAAAACCCGA AGGTCATTACAATTGGCACCATGGAGCAGTGCAGTACAGCGGAGG AAGGTTTACCATCCCCACAGGCGCCGGCAAACCGGGAGATAGCGG TAGGCCTATTTTTGACAACAAAGGGCGAGTAGTGGCCATCGTGTTA GGCGGGGCCAACGAAGGTGCTCGCACTGCGCTGTCTGTGGTGACGT GGACAAAAGACATGGTCACTCGGGTAACGCCAGAAGGAACTGAAG AGTGGTCTGCCGCGCTGATGATGTGTATCCTTGCCAACACCTCTTTC CCCTGCTCATCACCTCCCTGCTACCCCTGCTGCTACGAAAAACAGC CAGAACAGACACTGCGGATGCTGGAAGACAATGTGAATAGACCAG GGTACTATGAGCTACTGGAAGCGTCCATGACATGCAGAAACAGATC ACGCCACCGCCGTAGTGTAACAGAGCACTTCAATGTGTATAAGGCT ACTAGACCGTACTTAGCGTATTGCGCTGACTGTGGGGACGGGTACT TCTGCTATAGCCCAGTTGCTATCGAGAAGATCCGAGATGAGGCGTC TGACGGCATGCTCAAGATCCAAGTCTCCGCCCAAATAGGTCTGGAC AAGGCAGGTACCCACGCCCACACGAAGATCCGATATATGGCTGGTC ATGATGTTCAGGAATCTAAGAGAGATTCCTTGAGGGTGTACACGTC CGCAGCGTGCTCTATACATGGGACGATGGGACACTTCATCGTCGCA CATTGTCCGCCAGGCGACTACCTCAAGGTTTCGTTCGAGGACGCAG AYTCACACGTGAAGGCATGTAAGGTCCAATACAAGCACGACCCATT GCCGGTGGGTAGAGAGAAGTTCGTGGTTAGACCCCACTTTGGCGTA GAGCTGCCATGCACCTCATACCAGCTGACAACAGCTCCCACCGACG AGGAGATCGACATGCACACACCGCCAGATATACCGGATCGCACCCT GCTATCACAGACGGCGGGCAACGTCAAAATAACAGCAGGCGGCAG GACTATCAGGTACAATTGTACCTGTGGCCGTGACAACGTAGGCACT ACCAGTACTGACAAGACCATCAACACATGCAAGATTGACCAATGCC ATGCTGCCGTTACCAGCCATGACAAATGGCAATTTACCTCTCCATTT GTTCCCAGGGCTGATCAGACAGCTAGGAGGGGCAAAGTGCATGTTC CATTCCCTTTGACTAACGTCACCTGCCGAGTGCCGTTGGCTCGAGCG CCGGATGTCACCTATGGTAAGAAGGAGGTGACCCTGAGATTACACC CAGATCATCCGACGCTCTTCTCCTATAGGAGTTTAGGAGCCGAACC GCACCCGTACGAGGAGTGGGTTGACAAGTTCTCTGAGCGCATCATC CCAGTGACGGAAGAAGGGATTGAGTACCAGTGGGGCAACAACCCG CCGGTCCGCCTATGGGCGCAACTGACGACCGAGGGCAAACCCCAT GGCTGGCCACATGAAATCATTCAGTACTATTATGGACTATACCCCG CCGCCACCATTGCCGCAGTATCCGGGGCGAGTCTGATGGCCCTCCT AACTCTAGCGGCCACATGCTGCATGCTGGCCACCGCGAGGAGAAA GTGCCTAACACCATACGCCTTGACGCCAGGAGCGGTGGTACCGTTG ACACTGGGGCTGCTTTGCTGCGCACCGAGGGCGAACGCAGCATCAT TCGCTGAGACTATGGCATATCTGTGGGACGAGAACAAAACCCTCTT TTGGATGGAATTCGCGGCCCCAGCCGCAGCGCTTGCTTTGCTGGCA TGCTGTATCAAAAGCCTGATCTGCTGTTGTAAGCCATTTTCTTTTTT AGTGTTACTGAGCCTGGGAGCCTCCGCAAAAGCTTACGAGCACACA GCCACAATTCCGAATGTGGTGGGGTTCCCGTATAAGGCTCACATTG AAAGGAATGGCTTCTCGCCCATGACTCTGCAGCTTGAAGTGGTGGA GACAAGCTTGGAACCCACACTTAACCTGGAGTACATTACCTGCGAA TACAAGACGGTGGTCCCTTCGCCATTCATCAAATGTTGCGGAACAT CAGAATGCTCATCCAAGGAGCAGCCAGACTACCAATGCAAGGTGT ACACGGGTGTATACCCATTCATGTGGGGTGGAGCCTACTGTTTCTG CGACTCCGAGAACACGCAGCTCAGCGAGGCCTATGTCGACAGGTCA GACGTTTGCAAACATGATCACGCATCGGCCTACAAGGCACACACGG CCTCTCTAAAAGCAACAATCAGGATCAGTTATGGCACCATCAACCA GACCACCGAGGCCTTCGTTAATGGTGAACACGCGGTCAACGTGGGC GGAAGCAAGTTCATCTTTGGACCGATCTCAACAGCTTGGTCACCGT TCGACAATAAAATTGTCGTGTATAAAGATGATGTCTACAACCAGGA CTTCCCACCCTACGGATCAGGCCAGCCGGGTAGATTCGGAGACATT CAGAGCAGGACAGTGGAGAGCAAAGACTTGTATGCCAACACGGCC CTAAAACTCTCAAGACCATCACCCGGGGTTGTGCATGTGCCATACA CGCAGACACCATCCGGATTTAAATATTGGCTGAAGGAGAAAGGATC TTCATTGAATACAAAGGCCCCTTTTGGCTGCAAGATAAAGACCAAT CCAGTCAGAGCCATGGATTGTGCAGTTGGCAGTATACCTGTGTCGA TGGACATACCTGACAGTGCATTCACACGAGTGGTAGATGCCCCGGC TGTAACAGACCTGAGCTGCCAGGTAGTGGTCTGTACACACTCCTCC GATTTCGGAGGAGTTGCCACATTGTCTTACAAAACGGACAAACCCG GCAAGTGCGCTGTCCACTCACATTCCAACGTCGCAACGTTGCAAGA GGCGACGGTGGATGTCAAGGAGGATGGCAAGGTCACAGTGCACTT TTCCACGGCGTCCGCCTCCCCGGCCTTCAAAGTGTCCGTCTGTGACG CAAAAACAACGTGCACGGCGGCGTGCGAGCCTCCAAAAGACCACA TCGTCCCTTATGGGGCGAGCCATAACAACCAGGTCTTTCCGGACAT GTCAGGAACTGCGATGACGTGGGTGCAGAGGCTGGCCAGTGGGTT AGGTGGGCTGGCTCTCATCGCGGTGGTTGTGCTGGTCTTGGTAACCT GCATAACAATGCGTCGGTAAGCTTTAGTTCAAAGGGCCATATAAAC CCCTGAATAGTAACAAAATATAAAAATTACAAAATATGTAGTTCAA AGGGCTATACTACCCCTGATTAGTAACAAAATAGAAAACCACAAA ATATGTAGTTAAGTATTATAAGATGTGTAGTTCAAAGGGCTATATC ACCCCTGATTAGTAACAAAATATAAAAACAAAAATATGTAGTTAAG TACTAACCAACAAGTAGACAAATAGATGCTAACCATATATATAACC AGCTATAGTATACTATATTTAGCTAAGCAGTTGCAGTAGTAAGAAT GTAGTTCAAAGGGCTATACAACCCCTGAATAGTAACAAAATACAAA AATACTAATAAAAATTTAAAAATCACTAGAAATCCAATCATTAAAT TATTAATTGGCTAGCCGAACTCTAAGGAGATGTAGGCGTCCGAACT CTGCGGAGATGTAGGACTAAATTCTGCCGAACCCCATAACACCGGG GACGTAGGCGTCTAATTTGTTTTTTAATATTTTACAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA SEQ ID NO: 11 MNYIPTQTFYGRRWRPRPAFRPWQVPMQPTPTMVTPMLQAPDLQAQ QMQQLISAVSALTTKQNVKAPKGQRKKKQQKPKEKKENQKKKPTQK KKQQQKPKPQAKKKKPGRRERMCMKIENDCIFEVKLDGKVTGYACL VGDKVMKPAHVKGTIDNPDLAKLTYKKSSKYDLECAQIPVHMKSDAS KYTHEKPEGHYNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGR VVAIVLGGANEGARTALSVVTWTKDMVTRVTPEGTEEWSAALMMCI LANTSFPCSSPPCYPCCYEKQPEQTLRMLEDNVNRPGYYELLEASMTC RNRSRHRRSVTEHFNVYKATRPYLAYCADCGDGYFCYSPVAIEKIRDE ASDGMLKIQVSAQIGLDKAGTHAHTKIRYMAGHDVQESKRDSLRVYT SAACSIHGTMGHFIVAHCPPGDYLKVSFEDADSHVKACKVQYKHDPL PVGREKFVVRPHFGVELPCTSYQLTTAPTDEEIDMHTPPDIPDRTLLSQ TAGNVKITAGGRTIRYNCTCGRDNVGTTSTDKTINTCKIDQCHAAVTS HDKWQFTSPFVPRADQTARRGKVHVPFPLTNVTCRVPLARAPDVTYG KKEVTLRLHPDHPTLFSYRSLGAEPHPYEEWVDKFSERIIPVTEEGIEYQ WGNNPPVRLWAQLTTEGKPHGWPHEIIQYYYGLYPAATIAAVSGASL MALLTLAATCCMLATARRKCLTPYALTPGAVVPLTLGLLCCAPRANA ASFAETMAYLWDENKTLFWMEFAAPAAALALLACCIKSLICCCKPFSF LVLLSLGASAKAYEHTATIPNVVGFPYKAHIERNGFSPMTLQLEVVETS LEPTLNLEYITCEYKTVVPSPFIKCCGTSECSSKEQPDYQCKVYTGVYP FMWGGAYCFCDSENTQLSEAYVDRSDVCKHDHASAYKAHTASLKAT IRISYGTINQTTEAFVNGEHAVNVGGSKFIFGPISTAWSPFDNKIVVYKD DVYNQDFPPYGSGQPGRFGDIQSRTVESKDLYANTALKLSRPSPGVVH VPYTQTPSGFKYWLKEKGSSLNTKAPFGCKIKTNPVRAMDCAVGSIPV SMDIPDSAFTRVVDAPAVTDLSCQVVVCTHSSDFGGVATLSYKTDKPG KCAVHSHSNVATLQEATVDVKEDGKVTVHFSTASASPAFKVSVCDAK TTCTAACEPPKDHIVPYGASHNNQVFPDMSGTAMTWVQRLASGLGGL ALIAVVVLVLVTCITMRR SEQ ID NO: 12 MKVTVDVEADSPFLKALQKAFPAFEVESQQVTPNDHANARAFSHLAT KLIEQEVPTNITILDVGSAPARRLMSDHSYHCICPMKSAEDPERLANYA RKLAKAAGEVLDKNVSGKITDLQDVMATPDLESPTFCLHTDETCRTR AEVAVYQDVYAVHAPTSLYHQAMKGVRTVYWIGFDTTPFMFEVLAG AYPTYSTNWADEQVLQARNIGLCATSLSEGHRGKLSIMRKKRLRPSDR VMFSVGSTLYIESRRLLKSWHLPSVFHLKGKNSFTCRCDTIVSCEGYV VKKITMSPGTYGKTVGYAVTHHAEGFLMCKVTDTVRGERVSFPVCTY VPATICDQMTGILATDVTPEDAQKLLVGLNQRIVVNGRTQRNTNTMK NYLLPVVAQAFSKWAREAKADMEDEKPLGTRERTLTCCCLWAFKSH KTHTMYKRPDTQTIVKVPSTFDSFVIPSLWSSSLSIGLRQRIKLLLGPKL SRDLPYSGDRNEAREAEKEAEETKEAELTREALPPLVGSNCADDVDRV DVEELTYRAGAGVVETPRNALRVTPQERDQLIGAYLILSPQAVLKSEK LTPIHPLAEQVTIMTHSGRSGRYPVDRYDGRVLVPTGAAIPVSEFQALS ESATMVYNEREFINRKLEHIALYGPALNTEEENYEKVRAERAEAEYVF DVDKRMCVKREEASGLVLVGDLINPPFHEFAYEGLKIRPATPFQTTVIG VFGVPGSGKSAIIKSVVTTRDLVASGKKENCQEIVNDVKKQRGLDVTA RTVDSILLNGCRRGVENLYVDEAFACHSGTLLALIAMVKPTGKVILCG DPKQCGFFNLMQLKVNFNHDICTQVLHKSISRRCTLPITAIVSTLHYQG KMRTTNLCSAPIQIDTTGTTKPAKGDIVLTCFRGWVKQLQIDYRGHEV MTAAASQGLTRKGVYAVRQKVNENPLYAPSSEHVNVLLTRTENRLV WKTLSGDPWIKVLTNIPKGDFSATLEEWQEEHDNIMNALRERSTAVDP FQNKAKVCWAKCLVQVLETAGIRMTAEEWDTVLAFREDRAYSPEVA LNEICTKYYGVDLDSGLFSAQSVSLYYENNHWDNRPGGRMYGFNREV ARKFEQRYPFLRGKMDSGLQVNVPERKVQPFNAECNILPSNRRLPHAL VTSYQQCQGERVEWLLKKLPGYHLLLVSEYNLALPHKRVFWIAPPHV SGADRIYDLDLGLPLNAGRYDLVFVNIHTEYRTHHYQQCVDHSMKLQ MLGGDSLHLLKPGGSLLIRAYGYADRVSEMVVTALARKFSAFRVLRP ACVTSNTEVFLLFTNFDNGRRAVTLHQANQRLSSMFACNGLHTAGCA PSYRVRRTDISGHAEEAVVNAANAKGTVGDGVCRAVARKWPDSFKG AATPVGTAKLVQANGMNVIHAVGPNFSTVTEAEGDRELAAAYRAVA GIINASNIKSVAIPLLSTGVFSGGKDRVMQSLNHLFTAMDTTDADVVIY CRDKAWEKKIQEAIDRRTAVELVSEDISLESDLIRVHPDSCLVGRKGYS ITDGKLIISYLEGTRFHQTAVDMAEISTLWPKLQDANEQICLYALGESM DSIRTKCPVEDADSSTPPKTVPCLCRYAMTAERVARLRMNNTKAIIVC SSFPLPKYRIEGVQKVKCDRVLIFDQTVPSLVSPRKYIPAAASTHADTV SLDSTVSTGSAWSFPSEATYETMEVVAEVHHSEPPVPPPRRRRAQVTM HHQELLEVSDMHTPIAARVEIPVYDTAVVVERVAIPCTSEYAKPIPAPR AARVVPVPAPRIQRASTYRVSPTPTPRVLRASVCSVTTSAGVEFPWAPE DLEVLTEPVHCKMREPVELPWEPEDVDIQFGDFETSDKIQFGDIDFDQF LGRAGAYIFSSDTGPGHLQQKSVRQHALPCEMLYVHEEERTYPPALDE AREKLLQAKMQMAPTEANKSRYQSRKVENMKAVIIDRLKDGARTYL TEQSEKIPTYVSKYPRPVYSPSVEDSLQNPEVAVAACNAFLEANYPTV ASYQITDEYDAYLDMVDGSESCLDRATFCPAKLRCYPKHHAYHQPQV RSAVPSPFQNTLQNVLAAATKRNCNVTQMRELPTLDSAVLNVECFKK FACNGEYWQEFICDNPIRITTENITTYVTRLKGPKAAALFAKTHNLVPL QEVPMDRFVVDMKRDVKVTPGTKHTEERPKVQVIQAAEPLATAYLCG IHRELVRRLKAVLAPNIHTLFDMSAEDFDAIIAAHFQPGDAVLETDIASF DKSQDDSLALTALMLLEDLGVDQELLDLIEAAFGEITSVHLPTGTRFKF GAMMKSGMFLTLFVNTLLNIVIACRVLREKLTNSVCAAFIGDDNIVHG VRSDPLMAERCASWVNMEVKIIDATMCEKPPYFCGGFILYDKVTGSA CRVADPLKRLFKLGKPLPAGDTQDEDRRRALKDETDRWARVGLKSEL EIALSSRYEVNGIGNIVRAMATLAKSLKNFKKLRGPIVHLYGGPK SEQ ID NO: 13 ATGGGCGGCGCATGAGAGAAGCCCACACCAATTACCTACCCAAAA ATGGAGAGAGTTCACGTTGACATCGAGGAAGACAGCCCATTCCTCA GAGCTTTGCAACGGAGCTTCCCGCAGTTTGAGGTAGAAGCCAAGCA GGTCACTGATAATGACCATGCTAATGCCAGAGCGTTTTCGCATCTG GCTTCAAAATTGATCGAAACGGAGGTGGACCCATCCGACACGATCC TTGACATTGGAAGTGCGCCCGCCCGCAGAATGTATTCTAAGCATAA GTATCATTGCATCTGTCCGATGAGATGCGCGGAAGATCCGGACAGA TTGTACAAGTATGCAACTAAGCTGAAGAAAAATTGCAAGGAAATA ACTGACAAGGAATTGGACAAGAAAATGAAGGAGCTCGCCGCCGTC ATGAGCGACCCTGACCTGGAAACTGAGACTATGTGCCTCCACGACG ATGAGTCGTGTCGCTACGAAGGGCAAGTCGCTGTTTACCAGGATGT ATACGCAGTTGACGGACCGACAAGTCTCTATCACCAAGCCAACAAG GGAGTTAGAGTCGCCTATTGGATAGGCTTTGACACCACCCCTTTTAT GTTTAAGAACTTGGCTGGAGCATATCCATCATACTCTACTAACTGG GCCGACGAAACCGTGTTAACGGCTCGTAATATAGGCCTATGCAGCT CCGACGTTATGGAGCGGTCACGCAGAGGAATGTCCATTCTTAGGAA GAAGTATTTGAAACCATCCAATAATGTCTTATTCTCTGTTGGCTCGA CCATCTACCACGAGAAGAGGGACTTACTGAGGAGCTGGCACCTGCC GTCTGTATTTCACTTACGTGGCAAGCAAAATTACACATGTCGGTGT GAGACTATAGTTAGTTGCGACGGGTACGTCGTTAAAAGAATAGCTA TCAGTCCAGGCCTGTATGGGAAGCCTTCAGGCTATGCTGCTACGAT GCACCGCGAGGGATTCTTGTGCTGCAAAGTGACAGACACATTGAAC GGGGAGAGGGTCTCTTTTCCCGTGTGCACGTATGTGCCAGCTACAT TGTGTGACCAAATGACTGGCATACTGGCAACAGATGTCAGTGCGGA CGACGCGCAAAAACTGCTGGTTGGGCTCAACCAGCGCATAGTCGTC AACGGTCGCACCCAAAGAAATACCAATACCATGAAGAATTATCTTT TGCCCGTAGTGGCCCAGGCATTTGCTAGGTGGGCAAAGGAATATAA GGAAGATCAAGAAGATGAGAGGCCACTAGGACTACGAGATAGACA GTTAGTCATGGGGTGCTGCTGGGCTTTTAGAAGGCATAAGATAACA TCTATTTATAAGCGCCCAGATACCCAAACCATCATCAAAGTGAACA GCGATTTCCACTCATTCGTGCTGCCCAGGATAGGCAGTAACACATT GGAGATCGGGCTGAGAACAAGAATCAGGAAAATGCTAGAAGAGCA CAAGGAGCCGTCACCTCTCATTACTGCCGAGGACATACAAGAAGCT AAGTGCGCAGCCGATGAGGCTAAGGAGGTGCGTGAAGCCGAGGAG TTGCGCGCCGCACTACCACCTTTGGCAGCTGATGTTGAGGAGCCCA CTCTGGAAGCCGATGTTGACTTGATGTTACAAGAGGCTGGGGCCGG CTCAGTGGAGACACCTCGTGGCTTGATAAAGGTTACCAGCTATGCC GGCGAGGACAAGATCGGCTCTTACGCAGTGCTTTCTCCGCAGGCTG TACTTAAGAGTGAGAAACTATCTTGCATTCACCCTCTCGCTGAACA AGTCATAGTGATAACACACTCTGGCCGAAAAGGGCGTTACGCCGTG GAACCCTACCATGGAAAAGTAGTGGTGCCAGAGGGACATGCAATA CCCGTCCAGGACTTTCAAGCTCTGAGTGAAAGTGCTACCATTGTGT ACAACGAACGAGAGTTCGTAAACAGGTATCTGCACCATATTGCCAC ACATGGAGGAGCGCTGAACACAGATGAAGAATATTACAAAACTGT CAAGCCCAGCGAGCACGATGGCGAATACCTGTATGACATCGACAG GAAACAGTGCGTCAAGAAAGAACTAGTCACTGGGCTAGGGCTTAC AGGCGAGCTGGTGGACCCTCCCTTCCATGAATTCGCCTACGAGAGT CTGAGAACACGACCGGCCGCTCCTTACCAAGTACCAACCATAGGGG TGTATGGCGTGCCGGGATCAGGCAAGTCTGGCATCATCAAAAGCGC AGTCACCAAAAAAGATCTGGTGGTGAGCGCCAAGAAAGAAAACTG CGCAGAAATAATAAGGGACGTCAAGAAAATGAGAGGGCTGGACGT CAATGCCAGAACTGTGGACTCAGTGCTCTTGAATGGATGCAAACAC CCCGTAGAGACCCTGTATATTGACGAAGCTTTTGCTTGTCATGCAG GCACTCTCAGAGCGCTCATAGCCATCATAAGACCTAAAAAGGCAGT GCTCTGCGGGGATCCAAAACAGTGCGGCTTTTTCAATATGATGTGT CTGAAAGTGCATTTTAACCACGAGATTTGCACGCAGGTCTTCCACA AAAGCATCTCTCGTCGTTGCACTAAATCTGTGACTTCGGTCGTCTCA ACCTTGTTTTACGACAAAAGAATGAGAACGACGAACCCGAAAGAG ACTAAGATTGAGATTGACACTACTGGCAGTACCAAACCAAAGCAG GACGATCTCATTCTCACTTGTTTTAGAGGGTGGGTGAAGCAGTTGC AAATAGATTACAAAGGCAACGAGATAATGACGGCAGCTGCCTCTC AAGGGCTGACCCGTAAAGGCGTGTATGCCGTTCGGTACAAGGTGAA TGAAAACCCCCTGTACGCACCCACCTCAGAACACGTGAACGTCCTA CTGACCCGCACGGAGGACCGTATCGTGTGGAAAACACTAGCCGGC GATCCATGGATAAAAACACTGACGGCCAAGTATCCTGGGAATTTCA CTGCCACGATAGAGGAATGGCAAGCAGAGCATGATGCCATCATGA GGCACATCTTGGAGAGACCGGACCCTACCGATGTTTTCCAAAATAA GGCGAACGTGTGTTGGGCCAAGGCTTTGGTGCCGGTACTGAAGACT GCAGGCATAGACATGACCACTGAACAATGGAACACTGTGGATTATT TCGAAACGGACAAAGCTCACTCAGCAGAGATAGTGTTGAACCAACT ATGCGTGAGGTTCTTTGGACTCGACCTGGACTCCGGTCTATTTTCTG CACCCACTGTTCCGCTGTCCATTAGGAATAATCACTGGGATAATTCC CCGTCGCCTAACATGTACGGGCTGAATAAAGAAGTGGTCCGTCAGC TCTCCCGCCGGTACCCACAACTGCCTCGAGCAGTTGCCACTGGAAG AGTCTATGACATGAACACTGGTACACTGCGCAATTATGATCCGCGC ATAAATCTAGTACCTGTGAACAGAAGACTGCCTCATGCTTTAGTCC TCCACCATAATGAACACCCGCAGAGTGATTTTTCTTCATTCGTCAGC AAACTGAAGGGCAGAACTGTCTTGGTGGTCGGGGAGAAGTTGTCCG TCCCAGGCAAAACGGTTGATTGGTTGTCAGACAAGCCTGAGGCTAC CTTCAGAGCTCGGCTGGATTTAGGTATCCCAGGTGACGTGCCCAAA TACGACATTATATTTATTAACGTGAGGACTCCATATAAATACCATC ATTATCAGCAGTGTGAAGACCACGCCATTAAGCTTAGTATGTTGAC CAAGAAAGCTTGTCTGCATCTGAATCCCGGCGGAACCTGCGTCAGC ATAGGTTATGGTTACGCTGACAGGGCCAGCGAAAGCATCATTGGTG CTATAGCGCGGCAGTTCAAGTTCTCCCGGGTATGCAAACCTAAATC CTCACTTGAAGAGACAGAAGTTCTGTTTGTATTCATTGGGTACGATC GCAAGGCCCGTACGCATAATCCTTACAAGCTTTCATCTACCTTGACC AACATCTATACAGGTTCCAGACTCCACGAAGCCGGATGCGCACCCT CATATCATGTGGTGCGAGGGGATATTGCCATGGCCACCGAAGGAGT GATCATAAATGCCGCTAACAGCAAAGGACAACCTGGCGGAGGGGT GTGCGGAGCGCTGTATAAGAAATTCCCGGAAAGCTTCGATTTACAG CCAATCGAAGTGGGAAAAGCGCGACTGGTCAAAGGTGCAGCTAAA CACATCATTCATGCCGTAGGGCCCAACTTCAACAAAGTTTCGGAAG TTGAAGGTGACAAACAGTTGGCAGAGGCTTATGAGTCCATCGCTAA AATTGTCAACGATAACAATTACAAGTCAGTAGCGATTCCACTGTTG TCTACCGGCATCTTTTCTGGTAACAAAGATCGACTAACCCAATCATT GAACCATTTGCTGACAGCTTTAGACACCACTGATGCAGATGTAGCC ATATACTGCAGGGACAAGAAATGGGAAATGACTCTCAAGGAAGCA GTGGCTAGGAGAGAAGCAGTGGAGGAGATATGCATATCCGACGAC TCTTCGGTGACAGAACCGGATGCAGAGCTGGTGAGGGTACATCCGA AGAGTTCTTTGGCTGGCAGGAAGGGCTACAGCACAAGTGATGGCA AGACTTTCTCATATTTGGAAGGGACTAAATTCCACCAGGCGGCCAA GGATATAGCAGAAATTAATGCCATGTGGCCTGTTGCAACGGAGGCC AATGAGCAAGTATGCATGTATATCCTCGGTGAAAGCATGAGCAGCA TTAGGTCAAAATGCCCCGTCGAAGAATCGGAAGCCTCCACACCACC TAGCACGCTGCCTTGCTTGTGCATCCATGCTATGACTCCAGAAAGA GTACAACGCCTAAAAGCCTCGCGTCCAGAACAAATTACTGTGTGCT CATCCTTTCCATTGCCGAAGTATAGAATCACTGGTGTGCAGAAGAT CCAGTGCTCCCAGCCTATACTGTTCTCACCGAAGGTGCCTGCGTAC ATTCATCCACGGAAGTACCTCGTGGAAACACCACCGGTGGAAGAG ATTCCGGAGCTGCCGGCGGAGAACCAATCCACAGAGGGGACATCT GAACAACCAGCACCAAACGTGGATGCAACCAGGACTAGAACGCCT GAACCGATCATCATTGAAGAGGAAGAAGAGGATAGTATAAGTTTG CTGTCAGACGGCCCGACCCACCAGGTGCTGCAAGTCGAGGCAGAC ATCCACGGGCCGCCTTCTGTATCCAGCTCATCCTGGTCCATTCCTCA CGCATCCGACTTTGATGTGGACAGTTTATCCATCCTTGCCCTGGAGG GAGCTAGCGTGACTAGCGAGGCAGCGTCAGCCGAGACTAACTCAT ACTTCGCAAGGAGCATGGAGTTTCTGGCGCGACCGGTGCCTGCGCC TCGAACAGTATTCAGGAACCCTCCACATCCCGCTCCACGCACAAGA ACACCGTCACTTGCACCCAGCAGGGCCAGCTCGAGAACTAGCCTGG TTTCCACCCCGCCAGGCGTGAATAGGGTGATTACTAGAGAGGAGCT CGAGGCGCTTACCCCGTCCCGCGCTCCTAGCAGGTCGGCCTCAAGA ACTAGTCTGGTCTCTAACCCGCCAGGCGTAAATAGGGTGATTACAA GAGAGGAGTTTGAGGCGTTCGTGGCACAACAACAATGACGGTTTGA CGCGGGTGCATACATCTTTTCCTCCGATACCGGTCAAGGGCATTTAC AACAAAAATCAGTAAGGCAAACGGTGCTATCCGAAGTGGTTTTGGA GAGGACCGAATTGGAGATTTCGTATGCCCCGCGCCTCGACCAAGAA AAAGAAGAACTACTACGCAAGAAATTACAGCTGAATCCCACACCT GCTAACAGAAGCAGATACCAGTCCAGAAGGGTGGAGAACATGAAA GCCATAACAGCTAGACGTATTCTGCAAGGCCTAGGACATTATTTGA AGGCAGAAGGAAAAGTGGAGTGCTATCGAACCCTGCATCCTGTTCC TTTGTATTCATCTAGTGTGAATCGTGCTTTTTCAAGCCCCAAGGTCG CAGTGGAAGCCTGCAATGCCATGCTGAAAGAAAACTTTCCGACTGT AGCTTCTTACTGTATAATTCCAGAGTACGATGCCTATCTGGACATGG TTGACGGCGCTTCTTGTTGCTTAGACACTGCCAGTTTTTGCCCTGCG AAGCTGCGCAGTTTTCCAAAGAAACACTCTTACTTGGAACCCACAA TACGGTCGGCAGTGCCATCGGCGATTCAGAACACGCTCCAGAATGT CCTGGCAGCTGCCACAAAAAGAAATTGCAACGTCACGCAAATGAG AGAATTGCCCGTATTGGACTCGGCTGCCTTTAATGTGGAATGCTTCA AGAAATATGCGTGCAATAATGAATATTGGGAAACGTTTAAAGAAA ACCCCATCAGGCTTACTGAAGAAAATGTGGTAAATTACATTACTAA ATTAAAAGGACCAAAAGCTGCTGCTCTTTTTGCGAAGACACATAAT TTAAATATGTTACAGGACATACCAATGGACAGGTTTGTAATGGACT TAAAGAGGGACGTGAAAGTGACTCCAGGAACAAAACATACTGAAG AACGACCCAAGGTACAGGTGATCCAGGCCGCCGATCCGCTAGCGA CAGCGTATCTGTGCGGAATCCACCGGGAGTTGGTTAGGAGATTAAA TGCTGTCCTGCTTCCGAACATCCATACACTGTTTGACATGTCGGCTG AAGACTTTGACGCTATTATTGCCGAGCATTTCCAGCCTGGGGACTG TGTTCTGGAAACTGACATTGCGTCGTTTGATAAAAGTGAGGACGAC GCCATGGCTCTGACCGCGTTGATGATTCTGGAAGATCTAGGAGTGG ACGCAGAGCTGTTGACGCTGATTGAGGCGGCTTTCGGCGAAATATC ATCAATACATTTGCCCACCAAAACTAAATTTAAATTCGGAGCCATG ATGAAATCCGGAATGTTCCTCACACTGTTTGTGAACACAGTCATTA ACATCGTAATCGCAAGCAGAGTGTTAAGAGAGCGGCTAACCGGAT CACCATGTGCAGCATTCATTGGAGATGACAATATCGTGAAAGGAGT CAAATCTGACAAATTAATGGCAGACAGGTGCGCCACTTGGTTGAAC ATGGAAGTCAAGATCATAGACGCCGTGGTGGGCGAGAAAGCGCCC TATTTCTGTGGAGGGTTTATTTTGTGTGACTCCGTGACCGGCACAGC GTGCCGTGTGGCAGACCCCCTAAAAAGGCTGTTTAAGCTTGGCAAA CCTCTGGCAGCAGACGATGAACATGACGATGACAGGAGAAGGGCA TTATACGAAGAGTCAACACGCTGGAATCGAGTGGGAATTCTTCCAG AGCTGTGTAAGGCAGTAGAATCAAGGTATGAAACCGTAGGAACTTC CATCATAGTTATGGCCATGACTACTCTAGCTAGCAGTGTTAAGTCGT TCAGCTACCTGAGAGGGGCCCCTATAACTCTCTACGGCTAACCTGA ATGGACTACGACATAGTCTAGTCCGCCAAGATGTTCCCGTTCCAAC CAATGTATCCGATGCAGCCAATGCCCTATCGTAACCCGTTCGCGGC CCCGCGCAGGCCCTGGTTCCCCAGAACCGATCCTTTTCTGGCGATG CAGGTGCAGGAATTAACCCGCTCGATGGCTAACCTGACGTTCAAGC AACGCCGGGATGCGCCACCTGAAGGGCCACCCGCTAAGAAACCGA AGCGGGAGGCCCCGCAAAAACAAAAAGGGGGAGGCCAAGGGAAG AAGAAGAAGAATCAGGGGAAGAAGAAGGCTAAGACGGGGCCACC TAATCCGAAGGCACAGAGTGGAAACAAGAAGAAGACCAACAAGAA ACCAGGCAAGAGACAGCGCATGGTCATGAAATTGGAATCTGACAA GACATTCCCAATTATGCTGGAAGGGAAGATTAACGGCTACGCTTGC GTGGTCGGAGGGAAGTTATTCAGGCCGATGCACGTGGAAGGCAAG ATCGACAATGACGTTCTGGCCGCACTTAAGACGAAGAAAGCATCCA AATATGATCTTGAGTATGCAGATGTGCCACAGAACATGCGGGCCGA TACATTCAAGTACACCCACGAGAAGCCCCAAGGCTATTATAGCTGG CATCATGGAGCAGTCCAATATGAAAATGGGCGTTTCACGGTGCCAA AAGGAGTTGGGGCCAAGGGAGACAGCGGACGACCCATTCTGGACA ATCAGGGACGGGTGGTCGCTATTGTGCTGGGAGGTGTGAATGAAGG ATCTAGGACAGCCCTTTCAGTCGTCATGTGGAACGAGAAGGGAGTA ACTGTGAAGTATACTCCGGAGAACTGCGAGCAATGGTCACTAGTGA CCACCCTGTGTCTGCTCGCCAATGTGACGTTCCCATGTGCCCAACCA CCAATTTGCTACGACAGAAAACCAGCAGAGACCTTGGCCATGCTCA GCGTTAACGTTGACAACCCGGGCTACGATGAGCTGCTGGAAGCAGC TGTTAGGTGCCCCGGAAGAAAAAGGAGATCTACCGAGGAGCTGTTT AAGGAGTATAAGCTAACGCGCCCTTACATGGCCAGATGCATCAGAT GTGCCGTTGGGAGCTGCCATAGTCCAATAGCAATTGAGGCAGTGAA GAGCGACGGGCACGACGGCTATGTTAGACTTCAGACTTCCTCGCAG TACGGCCTGGATTCCTCTGGCAACTTAAAGGGAAGGACCATGCGGT ATGACATGCACGGAACCATTGAAGAGATACCGCTACATCAAGTGTC ACTCCACACATCTCGCCCGTGTCACATTGTGGATGGGCATGGTTATT TTCTGCTTGCTAGGTGCCCGGCAGGGGACTCCATCACTATGGAATTT AAGAAAGATTCAGTCACACACTCCTGCTCAGTGCCGTATGAAGTGA AATTTAATCCTGTAGGCAGAGAACTCTACACTCATCCCCCAGAACA CGGAGCAGAGCAAGCGTGCCAAGTCTACGCGCATGATGCACAGAA CAGAGGAGCTTATGTCGAGATGCACCTCCCGGGCTCAGAAGTGGAC AGCAGTTTGGTTTCCTTGAGCGGCAGTTCAGTCACCGTGACACCTCC TGCTGGGACTAGCGCCCTGGTGGAATGCGAGTGCGGCGGCACAAA GATCTCCGAGACCATCAACACGGCAAAACAGTTCAGCCAGTGCACA AAGAAGGAGCAATGCAGAGCATATCGACTGCAGAATGACAAATGG GTGTATAATTCTGACAAACTGCCCAAAGCAGCGGGAGCCACCCTAA AAGGAAAACTACACGTCCCATTTTTGCTGGCAGACGGCAAATGCAC CGTGCCTCTAGCACCGGAACCTATGATAACCTTCGGTTTCCGATCA GTGTCACTGAAACTGCACCCTAAGAATCCCACATATCTGACCACTC GCCAACTTGCTGATGAGCCTCATTACACGCACGAGCTCATATCTGA ACCAGCTGTTAGGAATTTTACCGTCACTGAAAAGGGGTGGGAGTTT GTATGGGGAAACCACCCGCCGAAAAGGTTTTGGGCACAGGAAACA GCACCCGGAAATCCACATGGGCTGCCACACGAGGTGATAACTCATT ATTACCACAGATACCCTATGTCCACCATCCTGGGTTTATCAATTTGC GCCGCCATTGTAACCGTTTCCGTTGCAGCGTCTACCTGGCTGTTCTG CAAATCCAGAGTTTCGTGCCTAACTCCTTACCGGCTAACACCTAAC GCCAGGATGCCGCTTTGCCTGGCTGTGCTTTGCTGCGCTCGCACTGC CCGGGCCGAGACCACCTGGGAGTCCTTGGATCACCTATGGAACAAT AACCAGCAGATGTTCTGGATTCAATTGCTGATCCCCCTGGCCGCCTT GATTGTAGTGACTCGCCTGCTCAAGTGCGTGTGCTGTGTAGTGCCTT TTTTAGTCGTGGCCGGCGCCGCAGGCGCCGGCGCCTACGAGCACGC GACCACGATGCCGAGCCAAGTGGGAATCTCGTATAACACCATAGTC AACAGAGCAGGCTACGCGCCACTCCCTATCAGCATAACACCAACA AAGATCAAGCTGATACCTACAGTGAACTTGGAGTACATCACCTGCC ACTACAAAACAGGAATGGATTCACCAGCCATTAAGTGCTGCGGATC TCAGGAATGTACTCCAACTTACAGGCCTGATGAACAGTGCAAAGTC TTCACTGGGGTTTACCCGTTCATGTGGGGAGGCGCATATTGCTTTTG CGACACTGAGAACACCCAAGTCAGCAAGGCCTACGTAATGAAATCT GACGACTGCCTTGCTGATCACGCTGAAGCATACAAAGCGCACACAG CCTCAGTGCAGGCGTTCCTCAACATCACAGTGGGAGAACACTCTAT TGTGACCACCGTGTATGTGAATGGAGAAACTCCTGTGAACTTCAAT GGGGTCAAATTAACTGCAGGTCCACTTTCCACAGCTTGGACACCCT TTGACCGCAAAATCGTGCAGTATGCCGGGGAGATCTATAATTACGA TTTTCCTGAGTACGGGGCAGGACAACCAGGAGCATTTGGAGACATA CAATCCAGAACAGTCTCGAGCTCAGATCTGTATGCCAATACCAACC TAGTGCTGCAGAGACCCAAAGCAGGAGCGATCCATGTGCCATACAC TCAGGCACCATCGGGTTTTGAGCAATGGAAGAAAGATAAAGCTCCG TCATTGAAATTCACCGCCCCTTTCGGATGCGAAATATATACAAACC CCATTCGCGCCGAAAATTGTGCCGTAGGGTCAATTCCATTAGCCTTT GACATCCCCGACGCCTTGTTCACCAGGGTGTCAGACACACCGACAC TTTCAGCGGCCGAATGCACTCTTAACGAGTGCGTGTATTCATCCGA CTTTGGCGGGATCGCCACGGTCAAGTATTCGGCCAGCAAGTCAGGC AAGTGCGCAGTCCATGTGCCATCAGGGACTGCTACCCTAAAAGAAG CAGCAGTCGAGTTAACCGAGCAAGGGTCGGTGACCATTCATTTCTC GACCGCAAATATCCACCCGGAGTTCAGGCTCCAAATATGCACATCA TATGTCATGTGCAAAGGTGATTGTCACCCCCCGAAAGACCACATTG TGACACACCCACAGTATCACGCCCAAACATTTACAGCCGCGGTGTC AAAAACCGCGTGGACGTGGTTAACATCCCTGCTGGGAGGATCGGCC GTAATTATTATAATTGGCTTAGTGCTGGCTACTATTGTGGCCATGTA CGTGCTGACCAACCAGAAACATAATTGAACATAGCAGCAATTGGCA AGCTGCTTATATAGAACTCGCGGCGATTGGCATGCCGCTTTAAAAT TTTATTTTATTTTCTTTTCTTTTC SEQ ID NO: 14 MFPFQPMYPMQPMPYRNPFAAPRRPWFPRTDPFLAMQVQELTRSMAN LTFKQRRDAPPEGPPAKKPKREAPQKQKGGGQGKKKKNQGKKKAKT GPPNPKAQSGNKKKTNKKPGKRQRMVMKLESDKTFPIMLEGKINGYA CVVGGKLFRPMHVEGKIDNDVLAALKTKKASKYDLEYADVPQNMRA DTFKYTHEKPQGYYSWHHGAVQYENGRFTVPKGVGAKGDSGRPILD NQGRVVAIVLGGVNEGSRTALSVVMWNEKGVTVKYTPENCEQWSLV TTLCLLANVTFPCAQPPICYDRKPAETLAMLSVNVDNPGYDELLEAAV RCPGRKRRSTEELFKEYKLTRPYMARCIRCAVGSCHSPIAIEAVKSDGH DGYVRLQTSSQYGLDSSGNLKGRTMRYDMHGTIEEIPLHQVSLHTSRP CHIVDGHGYFLLARCPAGDSITMEFKKDSVTHSCSVPYEVKFNPVGRE LYTHPPEHGAEQACQVYAHDAQNRGAYVEMHLPGSEVDSSLVSLSGS SVTVTPPAGTSALVECECGGTKISETINTAKQFSQCTKKEQCRAYRLQN DKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGF RSVSLKLHPKNPTYLTTRQLADEPHYTHELISEPAVRNFTVTEKGWEF VWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMSTILGLSICAA IVTVSVAASTWLFCKSRVSCLTPYRLTPNARMPLCLAVLCCARTARAE TTWESLDHLWNNNQQMFWIQLLIPLAALIVVTRLLKCVCCVVPFLVV AGAAGAGAYEHATTMPSQVGISYNTIVNRAGYAPLPISITPTKIKLIPTV NLEYITCHYKTGMDSPAIKCCGSQECTPTYRPDEQCKVFTGVYPFMW GGAYCFCDTENTQVSKAYVMKSDDCLADHAEAYKAHTASVQAFLNI TVGEHSIVTTVYVNGETPVNFNGVKLTAGPLSTAWTPFDRKIVQYAGE IYNYDFPEYGAGQPGAFGDIQSRTVSSSDLYANTNLVLQRPKAGAIHV PYTQAPSGFEQWKKDKAPSLKFTAPFGCEIYTNPIRAENCAVGSIPLAF DIPDALFTRVSDTPTLSAAECTLNECVYSSDFGGIATVKYSASKSGKCA VHVPSGTATLKEAAVELTEQGSVTIHYSTANIHPEFRLQICTSYVMCKG DCHPPKDHIVTHPQYHAQTFTAAVSKTAWTWLTSLLGGSAVIIIIGLVL ATIVAMYVLTNQKHN SEQ ID NO: 15 MERVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLAS KLIETEVDPSDTILDIGSAPARRMYSKHKYHCICPMRCAEDPDRLYKYA TKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYE GQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAG AYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNN VLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYV VKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYV PATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKN YLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHK ITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPS PLITAEDIQEAKCAADEAKEVREAEELRAALPPLAADVEEPTLEADVD LMLQEAGAGSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCI HPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESA TIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDID RKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVY GVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMRGLDVNART VDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQ CGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKRMR TTNPKETKIEIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAA ASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLA GDPWIKTLTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKA NVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLC VRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSR RYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHN EHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKTVDWLSDKPEATFRARL DLGIPGDVPKYDIIFINVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLN PGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSLEETEVLFVFI GYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIAMATE GVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKH IIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIF SGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARRE AVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGT KFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESE ASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKI QCSQPILFSPKVPAYIHPRKYLVETPPVEEIPELPAENQSTEGTSEQPAPN VDATRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSW SIPHASDFDVDSLSILALEGASVTSEAASAETNSYFARSMEFLARPVPAP RTVFRNPPHPAPRTRIPSLAPSRASSRTSLVSTPPGVNRVITREELEALT PSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQRRFDAGAYIFSS DTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQ LNPTPANRSRYQSRRVENMKAITARRILQGLGHYLKAEGKVECYRTLH PVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLD MVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVL AAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPI RLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRD VKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRLNAVLLPN IHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMI LEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVN TVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWL NMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKP LAADDEHDDDRRRALYEESTRWNRVGILPELCKAVESRYETVGTSIIV MAMTTLASSVKSFSYLRGAPITLYG SEQ ID NO: 16 TAGAGGCAACCACCCTATTTCCACCTATCCAAAATGGAGAAAGTTC ATGTTGACTTAGACGCAGACAGCCCATTCGTCAAGTCACTGCAAAG ATGCTTTCCACATTTTGAGATAGAAGCAACGCAGGTCACTGACAAT GACCATGCTAATGCTAGGGCGTTTTCGCACCTAGCTACTAAGCTCA TTGAGGGAGAAGTGGATACAGACCAGGTGATCCTGGATATTGGGA GCGCGCCTGTAAGGCACACGCATTCCAAACATAAGTACCACTGTAT TTGCCCAATGAAGAGCGCAGAAGACCCTGACAGACTCTACCGCTAC GCAGACAAGCTTAGAAAGAGTGATGTCACTGACAAATGTATTGCCT CTAAGGCCGCGGACCTGCTAACAGTAATGTCGACGCCTGACGCTGA GACACCCTCGTTATGCATGCACACTGACTCAACTTGCAGGTACCAC GGCTCCGTGGCCGTATATCAGGATGTATATGCAGTGCATGCACCGA CTTCCATTTACTACCAGGCGCTGAAAGGTGTACGAACTATCTATTG GATCGGGTTCGATACTACACCGTTCATGTATAAGAACATGGCAGGC GCCTACCCTACATACAACACTAATTGGGCCGATGAAAGTGTGTTGG AAGCCAGAAATATAGGGCTGGGTAGTTCAGACTTGCACGAAAAGA GTTTCGGAAAAGTATCCATTATGAGGAAGAAGAAATTACAACCCAC CAATAAAGTAATATTTTCTGTGGGGTCAACTATTTATACTGAAGAG AGAATACTGTTACGCAGTTGGCATCTACCTAATGTTTTTCATCTAAA AGGTAAAACTAGCTTTACAGGCAGATGTAATACTATCGTCAGCTGC GAAGGTTACGTTGTCAAGAAGATTACGCTCAGTCCTGGGATTTACG GGAAAGTGGATAATCTTGCTTCGACCATGCACCGAGAGGGATTCTT AAGTTGCAAGGTTACAGATACGTTAAGAGGGGAGAGGGTCTCTTTT CCCGTGTGTACGTACGTGCCAGCGACACTGTGCGACCAGATGACCG GGATACTGGCGACTGACGTCAGTGTCGATGACGCCCAGAAGCTGCT GGTTGGGCTCAACCAGCGAATTGTCGTCAATGGCAGAACACAACGT AACACAAATACCATGCAGAATTATCTATTACCAGTGGTCGCCCAGG CGTTCTCGCGGTGGGCGCGGGAACACCGCGCAGACCTGGAGGACG AAAAAGGGCTAGGGGTACGGGAACGTTCCCTAGTCATGGGCTGCTG CTGGGCTTTCAAAACTCACAAGATCACATCCATTTACAAGAGACCT GGGACTCAAACTATCAAGAAGGTGCCCGCCGTATTCAATTCCTTTG TCATCCCACAACCAACCAGCTATGGGCTTGATATAGGGTTGCGTCG CCGCATTAAGATGCTATTCGACGCAAAGAAGGCACCCGCTCCAATT ATTACTGAGGCCGACGTTGCACACCTTAAAGGCCTGCAGGATGAAG CTGAAGCCGTGGCAGAGGCTGAAGCCGTACGTGCAGCACTACCACC ACTTCTGCCGGAGGTCGACAAGGAGACCGTAGAGGCTGACATCGA CCTGATCATGCAGGAGGCAGGAGCAGGTAGCGTGGAGACACCTAG ACGACACATCAAGGTCACGACGTACCCAGGAGAAGAAATGATCGG CTCGTACGCAGTGCTTTCACCACAAGCGGTCCTTAACAGCGAGAAG CTCGCTTGTATTCACCCGTTAGCTGAGCAAGTGCTCGTGATGACTCA CAAGGGGCGCGCAGGACGATACAAGGTAGAGCCATACCACGGTAG AGTTATCGTCCCTAGTGGTACAGCTATACCAATCCCCGATTTCCAGG CTCTGAGTGAAAGTGCAACCATAGTATTTAACGAACGGGAGTTCGT TAACCGTTACTTACACCACATTGCCGTTAACGGAGGGGCACTGAAT ACAGATGAAGAGTACTACAAGGTTGTGAAAAGCACTGAGACAGAC TCTGAGTACGTATTTGACATCGACGCAAAGAAGTGCGTGAAGAAAG GGGATGCCGGACCAATGTGCCTGGTCGGCGAATTAGTAGACCCGCC ATTCCACGAATTCGCGTACGAGAGTTTAAAAACACGTCCTGCTGCA CCACACAAAGTGCCTACCATCGGAGTTTATGGAGTCCCAGGTTCCG GAAAGTCTGGTATAATCAAAAGCGCTGTTACCAAACGTGATCTGGT GGTCAGTGCAAAGAAAGAAAATTGCATGGAAATCATTAAAGACGT CAAACGTATGCGCGGCATGGACATCGCCGCCCGCACAGTGGATTCG GTGCTGCTAAATGGGGTAAAACACTCCGTCGACACACTGTACATAG ACGAGGCATTCGCTTGCCATGCAGGGACCCTGCTAGCACTTATTGC CATCGTCAAGCCAAAGAAAGTTGTATTGTGTGGAGATCCGAAACAA TGCGGCTTCTTTAACATGATGTGTCTAAAAGTGCATTTTAACCACGA GATATGCACAGAAGTGTATCACAAGAGTATTTCTCGGCGATGCACT AAGACAGTGACATCCATCGTTTCCACCCTGTTCTATGATAAACGCA TGAGAACTGTCAACCCATGCAATGATAAGATCATAATAGATACCAC CAGTACTACCAAACCTTTAAAGGATGACATAATATTAACCTGCTTT AGAGGGTGGGTTAAACAACTGCAGATTGACTACAAGAACCACGAG ATCATGACTGCAGCGGCCTCACAGGGGCTTACTAGAAAAGGGGTAT ACGCAGTGCGCTACAAGGTCAATGAGAACCCACTATACGCACAGA CATCTGAGCATGTGAATGTATTACTTACACGCACTGAAAAACGTAT AGTATGGAAGACTTTGGCCGGTGACCCTTGGATCAAGACGTTGACA GCATCGTATCCGGGTAATTTCACCGCCACACTGGAAGAATGGCAAG CTGAGCATGACGCTATCATGGCGAAAATACTTGAGACACCAGCTAG CAGCGACGTTTTCCAAAATAAAGTGAACGTCTGCTGGGCCAAAGCG CTAGAACCTGTGTTGGCCACCGCCAATATTACGCTGACCCGCTCGC AGTGGGAGACCATTCCAGCGTTCAAGGATGACAAAGCGTATTCGCC TGAGATGGCCTTAAACTTTTTCTGCACCAGATTCTTTGGCGTCGACA TCGACAGCGGGTTGTTCTCCGCGCCAACTGTTCCGCTGACTTACACC AATGAACACTGGGATAATAGCCCAGGTCCAAACATGTATGGTTTGT GCATGCGCACTGCTAAAGAACTTGCACGTCGGTATCCTTGTATTCTG AAAGCCGTGGATACAGGTAGAGTGGCTGACGTTCGCACAGACACT ATCAAAGACTATAACCCGCTAATAAATGTGGTACCCTTGAATAGAA GACTCCCACACTCATTGGTTGTCACACATAGATACACTGGGAACGG TGATTACTCCCAGCTAGTGACCAAGATGACCGGAAAGACCGTACTA GTAGTGGGTACACCTATGAACATACCAGGAAAGAGAGTCGAGACA CTAGGCCCAAGCCCACAATGTACATATAAAGCGGAACTGGACCTGG GCATTCCTGCCGCTTTAGGCAAATATGACATCATTTTTATTAACGTG AGGACTCCCTACCGACACCACCATTACCAACAGTGCGAGGACCATG CGATCCACCACAGCATGCTTACCAGAAAAGCAGTGGACCATTTGAA CAAAGGCGGTACGTGCATCGCATTGGGCTATGGGACTGCGGACAG AGCCACCGAGAACATTATCTCTGCAGTCGCCCGCTCATTCAGGTTCT CACGTGTGTGCCAGCCGAAGTGTGCCTGGGAAAACACTGAGGTCGC GTTCGTGTTTTTCGGCAAGGACAACGGCAACCATCTCCAAGATCAA GATAGGCTGAGTGTTGTGCTAAACAACATATACCAAGGGTCAACTC AACATGAAGCTGGCAGAGCACCTGCGTACAGAGTGGTGCGCGGCG ACATAACAAAGAGCAATGATGAGGTTATTGTTAACGCGGCGAACA ACAAAGGGCAACCCGGTGGCGGTGTGTGTGGCGCCCTTTACAGGAA GTGGCCTGGAGCTTTTGATAAGCAGCCGGTAGCAACTGGTAAAGCG CACCTCGTCAAGCATTCTCCGAACGTCATCCATGCTGTTGGTCCTAA TTTCTCTCGGCTATCAGAAAACGAAGGAGACCAGAAATTGTCTGAA GTGTACATGGACATTGCCAGAATTATCAACAACGAGAGGTTTACTA AAGTCTCCATTCCGTTGTTATCTACCGGCATCTACGCAGGTGGTAAG GACAGGGTTATGCAATCGCTGAACCATTTATTTACAGCCATGGATA CTACCGACGCAGACATTACTATTTACTGTCTAGATAAGCAATGGGA GTCAAGAATAAAGGAAGCTATCACTCGGAAGGAAAGCGTTGAAGA GCTTACTGAGGATGACAGACCAGTTGACATTGAACTGGTACGGGTG CACCCGTTGAGCAGCTTGGCAGGTAGACCTGGTTATTCAACCACCG AGGGCAAGGTGTATTCGTATCTAGAGGGGACTAGGTTTCATCAAAC TGCCAAAGACATAGCTGAAATTTACGCTATGTGGCCTAACAAGCAA GAAGCAAACGAGCAGATTTGCTTATACGTGTTGGGAGAGAGTATGA ACAGCATCCGCTCTAAGTGTCCAGTTGAAGAGTCGGAGGCCTCTTC CCCCCCTCACACCATCCCGTGTCTGTGCAACTATGCAATGACTGCA GAGCGAGTTTACAGATTACGTATGGCAAAGAATGAACAATTCGCAG TTTGTTCGTCCTTTCAGTTACCGAAATACAGGATTACAGGGGTTCAG AAAATTCAATGCAGTAAACCTGTGATATTCTCTGGCACTGTACCCC CGGCCATACATCCAAGAAAATTCGCATCCGTGACAGTGGAAGACAC TCCGATGGTCCAACCTGAAAGGTTGGTGCCTAGGCGACCTGCACCG CCTGTGCCCGTACCTGCAAGAATCCCCAGCCCTCCATGTACATCGA CCAATGGATCGACGACCAGTATACAATCACTGGGGGAGGATCAAA GCGCATCTGCTTCTAGCGGAGCTGAAATCTCTGTAGACCAGGTTTC GCTATGGAGCATACCCAGCGCTACCGGGTTCGATGTGCGTACCTCC TCATCGTTGAGCCTAGAGCAGCCTACCTTTCCGACAATGGTTGTCG AAGCAGAGATTCACGCCAGTCAAGGATCACTGTGGAGCATACCCA GTATCACCGGATCTGAAACCCGTGCTCCGTCACCTCCAAGTCAGGA TAGTAGACCTTCCACCCCATCTGCAAGTGGTTCACACACGTCCGTG GACTTAATCACGTTTGACAGCGTTGCAGAGATTTTGGAGGATTTCA GTCGTTCGCCGTTTCAATTTTTGTCTGAAATCAAACCTATTCCTGCA CCTCGTACCCGAGTTACTAACATGAGCCGCAGCGCAGACACGATCA AACCAATTCCAAAGCCGCGTAAATGCCAGGTGAAGTACACGCAGC CACCTGGCGTCGCCAGGGCCATATCGGCAGCGGAATTTGACGAGTT TGTGCGGAGGCACTCGAATTGACGGTACGAAGCGGGTGCGTACATT TTCTCATCCGAGACGGGACAAGGGCACCTGCAACAAAAATCTACGC GGCAATGCAAACTCCAGTATCCAATCCTGGAGCGTTCCGTCCATGA GAAATTTTACGCCCCGCGCCTCGATCTCGAGCGTGAGAAGCTGTTG CAGAAGAAACTACAATTGTGTGCTTCTGAAGGTAATCGGAGCAGGT ATCAGTCTCGTAAAGTAGAGAACATGAAGGCAATCACCGTTGAGCG TCTACTGCAGGGGATAGGCTCATACCTCTCTGCAGAACCGCAACCA GTTGAATGCTACAAAGTCACCTATCCTGCTCCCATGTATTCAAGTAC TGCAAGCAACAGCTITTCATCAGCAGAAGTGGCCGTCAAAGTCTGC AACCTAGTACTGCAAGAGAATTTTCCCACCGTAGCCAGCTATAACA TAACGGATGAGTATGATGCCTATCTTGATATGGTGGACGGAGCATC CTGCTGTTTAGATACTGCCACCTTTTGCCCAGCCAAATTAAGGAGCT TTCCAAAGAAGCACAGTTATTTGCGGCCTGAGATACGGTCAGCAGT GCCATCACCGATTCAAAACACGCTCCAGAATGTACTAGCAGCAGCC ACGAAACGGAATTGCAATGTCACTCAAATGAGGGAACTTCCAGTGT TGGATTCAGCTGCCTTCAACGTGGAGTGTTTCAAAAAGTACGCCTG TAACGATGAGTACTGGGACTTCTACAAGACAAACCCGATAAGACTC ACCGCAGAAAATGTTACTCAGTATGTTACTAAGTTAAAGGGACCCA AAGCAGCTGCCCTTTTTGCGAAAACGCATAACTTACAGCCATTGCA TGAGATACCAATGGATAGATTCGTGATGGACCTTAAACGGGATGTT AAGGTTACACCCGGGACAAAACATACTGAAGAAAGACCAAAAGTT CAGGTGATACAGGCAGCTGATCCACTTGCAACCGCCTACCTATGTG GTATACATCGAGAGCTTGTGCGCAGGTTGAACGCAGTGCTGCTACC GAACATCCACACTTTGTTTGACATGTCTGCAGAAGATTTTGATGCTA TCATTGCCGAACACTTTCAATTCGGCGACTCGGTGTTAGAGACAGA CATAGCTTCTTTTGATAAAAGCGAGGACGATGCTATCGCCATGTCT GCTCTAATGATTCTTGAAGACCTAGGAGTTGATCAGGCACTGTTAA ACCTAATTGAAGCAGCCTTTGGGAACATAACATCTGTGCACTTACC AACAGGCACCCGATTTAAGTTCGGGGCAATGATGAAATCCGGGATG TTTTTGACACTCTTTATTAATACTGTTGTCAATATCATGATCGCTAG CCGCGTGCTCCGCGAGCGGTTGACCACTTCCCCCTGCGCAGCATTT ATCGGCGACGACAACATCGTGAAAGGGGTTACATCTGACGAGCTG ATGGCAGAGCGGTGCGCCACGTGGTTGAACATGGAAGTGAAGATC ATCGATGCAGTAGTCGGAGTAAAGGCACCGTACTTTTGCGGAGGGT TCATCGTAGTCGATCAGATCACAGGAACTGCGTGCAGAGTCGCCGA CCCCCTGAAGAGACTGTTTAAGCTAGGTAAGCCGCTTCCATTGGAC GATGACCAAGACGTCGACAGGCGCAGAGCTCTGCATGATGAAGCG GCACGTTGGAACAGAATTGGCATCACTGAAGAGCTGGTGAAAGCA GTTGAATCACGCTACGAGGTGAACTACGTGTCACTAATCATTACAG CGTTGACTACATTAGCATCTACAGTTAGCAACTTTAAACACATAAG AGGTCACCCCATAACCCTCTACGGCTGACCTAAATAGGTTGTGCAT TAGTACCTAACCTATTTATATTATATTGCTATCTAAATATCAGAGAT GTTCCCATACCCTACACTTAACTACCCGCCTATGGCGCCGATTAACC CGATGGCCTACCGGGATCCTAATCCGCCTAGGCGCAGGTGGCGGCC CTTTAGGCCACCACTTGCAGCTCAAATTGAGGACCTGAGACGTTCC ATTGCTAACCTGACTTTGAAACAACGAGCACCTAACCCTCCAGCAG GACCGCCCGCCAAACGCAAGAAGCCTGCGCCCAAGCCTAAGCCTG CGCAGGCGAAAAAGAAGCGACCACCACCACCTGCCAAGAAACAAA AACGTAAACCTAAACCAGGCAAACGACAGCGAATGTGTATGAAGC TAGAGTCAGATAAAACGTTTCCGATCATGTTGAACGGACAGGTGAA TGGTTACGCGTGCGTCGTGGGTGGACGAGTGTTTAAACCGCTGCAC GTAGAAGGCAGAATAGACAATGAGCAACTGGCCGCTATCAAGCTG AAGAAGGCCAGCATATATGACCTTGAGTACGGTGATGTGCCACAAT GCATGAAATCAGATACCCTCCAGTACACCAGTGACAAGCCTCCTGG CTTTTATAACTGGCATCATGGAGCTGTGCAGTATGAGAACAACAGG TTCACCGTACCACGAGGGGTAGGTGGAAAGGGCGACAGCGGGAGA CCTATTCTTGACAACAAAGGTAGAGTCGTCGCAATTGTCCTGGGTG GAGTCAACGAAGGATCCAGGACGGCTCTATCAGTGGTGACATGGA ACCAAAAGGGGGTTACAGTCAAAGATACACCAGAGGGGTCAGAGC CATGGTCGCTCGCCACTGTCATGTGCGTCCTGGCCAATATCACGTTT CCATGTGATCAACCACCCTGCATGCCATGCTGTTATGAAAAGAATC CACACGAAACACTCACCATGCTGGAACAGAATTACGACAGCCGAG CCTATGATCAGCTGCTCGATGCCGCTGTGAAATGTAATGCTAGGAG AACCAGGAGAGATTTGGACACTCATTTCACCCAGTATAAGTTGGCA CGCCCGTATATTGCTGATTGCCCTAACTGTGGGCATAGTCGGTGCG ACAGCCCTATAGCTATAGAAGAAGTCAGAGGGGATGCGCATGCAG GAGTCATCCGCATCCAGACATCAGCTATGTTTGGTCTGAAGACGGA TGGAGTCGATTTGGCCTACATGAGTTTCATGAACGGCAAAACGCAG AAATCAATAAAGATCGACAACCTGCATGTGCGCACCTCAGCCCCTT GTTCCCTCGTGTCGCACCACGGCTATTACATCTTGGCTCAATGCCCA CCAGGGGACACGGTTACAGTTGGGTTTCACGACGGGCCTAACCGCC ATACGTGCACAGTTGCCCATAAGGTAGAATTCAGGCCAGTGGGTAG AGAGAAATACCGTCACCCACCTGAACATGGAGTTGAATTACCGTGT AACCGTTACACTCACAAGCGTGCAGACCAAGGACACTATGTTGAGA TGCATCAACCAGGGCTAGTTGCCGACCACTCTCTCCTTAGCATCCAC AGTGCCAAGGTGAAAATTACGGTACCGAGCGGCGCCCAAGTGAAA TACTACTGCAAGTGTCCAGATGTACGAGAGGGAATTACCAGCAGCG ACCATACAACCACCTGCACGGATGTCAAACAATGCAGGGCTTACCT GATTGACAACAAGAAATGGGTGTACAACTCTGGAAGACTGCCTCGA GGAGAGGGCGACACTTTTAAAGGAAAACTTCATGTGCCCTTTGTGC CTGTTAAGGCCAAGTGCATCGCCACGCTGGCACCGGAGCCTCTAGT TGAGCACAAACACCGCACCCTGATTTTACACCTGCACCCGGACCAC CCGACCTTGCTGACGACCAGGTCACTTGGAAGTGATGCAAATCCAA CTCGACAATGGATTGAGCGACCAACAACTGTCAATTTCACAGTCAC CGGAGAAGGGTTGGAGTATACCTGGGGAAACCATCCACCAAAAAG AGTATGGGCTCAAGAGTCAGGAGAAGGGAACCCACATGGATGGCC GCACGAAGTGGTAGTCTATTACTACAACAGATACCCGCTAACCACA ATTATCGGGTTATGCACCTGTGTGGCTATCATCATGGTCTCTTGTGT CACATCCGTGTGGCTCCTTTGCAGGACTCGCAATCTTTGCATAACCC CGTATAAACTAGCCCCGAACGCTCAAGTCCCAATACTCCTGGCGTT ACTTTGCTGCATTAAGCCGACGAGGGCAGACGACACCTTGCAAGTG CTGAATTATCTGTGGAACAACAATCAAAACTTTTTCTGGATGCAGA CGCTTATCCCACTTGCAGCGCTTATCGTATGCATGCGCATGCTGCGT TGCTTATTTTGCTGTGGGCCGGCTTTTTTACTTGTCTGCGGCGCCTTG GGCGCCGCAGCGTACGAACACACAGCAGTGATGCCGAACAAGGTG GGGATCCCGTATAAAGCTTTAGTCGAACGCCCAGGGTATGCACCCG TTCACCTACAGATACAGCTGGTTAATACCAGGATAATTCCATCAAC TAACCTGGAGTACATCACCTGCAAGTACAAGACAAAAGTGCCGTCT CCAGTAGTGAAATGCTGCGGTGCCACTCAATGTACCTCTAAACCCC ATCCTGACTATCAGTGTCAGGTGTTTACAGGTGTTTACCCATTCATG TGGGGAGGAGCCTACTGCTTCTGCGACACCGAAAACACCCAGATGA GCGAGGCGTATGTAGAGCGCTCGGAAGAGTGCTCTATCGACCACGC AAAAGCTTATAAAGTACACACAGGCACTGTTCAGGCAATGGTGAAC ATAACTTATGGGAGCGTCAGCTGGAGATCTGCAGATGTCTACGTCA ATGGTGAAACTCCCGCGAAAATAGGAGATGCCAAACTCATCATAG GTCCACTGTCATCTGCGTGGTCCCCATTCGATAACAAGGTGGTGGTT TATGGGCATGAAGTGTATAATTACGACTTTCCTGAGTACAGCACCG GCAAAGCAGGCTCTTTCGGAGACCTGCAATCACGCACATCAACCAG CAACGATCTGTACGCAAATACCAACTTGAAGCTACAACGACCCCAG GCTGGTATCGTGCACACACCTTTCACCCAGGCGCCCTCTGGCTTCGA ACGATGGAAAAGGGACAAAGGGGCACCGTTGAACGACGTAGCCCC GTTTGGCTGTTCAATTGCCCTGGAGCCGCTCCGTGCAGAAAATTGT GCAGTGGGAAGCATCCCTATATCTATAGATATACCCGATGCGGCTT TCACCAGAATATCTGAAACACCGACAGTCTCAGACCTGGAATGCAA AATTACGGAGTGTACTTATGCCTCCGATTTCGGTGGTATAGCCACC GTTGCCTACAAATCCAGTAAAGCAGGAAACTGTCCAATTCATTCTC CATCGGGTGTTGCAGTTATTAAAGAGAATGACGTCACCCTTGCTGA GAGCGGATCATTTACATTCCACTTCTCCACTGCAAACATCCATCCTG CTTTTAAGCTGCAGGTCTGCACCAGTGCAGTTACCTGCAAAGGAGA TTGCAAGCCACCGAAAGATCATATCGTCGATTATCCAGCACAACAT ACCGAATCCTTTACGTCGGCGATATCCGCCACCGCGTGGTCGTGGC TAAAAGTGCTGGTAGGAGGAACATCAGCATTTATTGTTCTGGGGCT TATTGCTACAGCAGTGGTTGCCCTAGTTCTGTTCTTCCATAGACATT AACATCTTGTCAACCACATAACACTACAGGCAGTGTATAAGGCTGT CTTACTAAACACTAAAATCACCCTAGTTCGATGTACTTCCGAGCTAT GGTGACGGTGGTGCATAATGCCGCCGATGCAGTGCATAAGGCTGCT ATATTACCAAATTATAACACTAAGGGCAGTGCATAATGCTGCTCCT AAGTAATTTTATACACACTTTATAATCAGGCATAATTGCCGTATATA CAATTACACTACAGGTAATATACCGCCTCTTATAAACACTACAGGC AGCGCATAATGCTGTCTTTTATATCAATTTACAAAATCATAT SEQ ID NO: 17 MFPYPTLNYPPMAPINPMAYRDPNPPRRRWRPFRPPLAAQIEDLRRSIA NLTLKQRAPNPPAGPPAKRKKPAPKPKPAQAKKKRPPPPAKKQKRKP KPGKRQRMCMKLESDKTFPIMLNGQVNGYACVVGGRVFKPLHVEGRI DNEQLAAIKLKKASIYDLEYGDVPQCMKSDTLQYTSDKPPGFYNWHH GAVQYENNRFTVPRGVGGKGDSGRPILDNKGRVVAIVLGGVNEGSRT ALSVVTWNQKGVTVKDTPEGSEPWSLATVMCVLANITFPCDQPPCMP CCYEKNPHETLTMLEQNYDSRAYDQLLDAAVKCNARRTRRDLDTHFT QYKLARPYIADCPNCGHSRCDSPIAIEEVRGDAHAGVIRIQTSAMFGLK TDGVDLAYMSFMNGKTQKSIKIDNLHVRTSAPCSLVSHHGYYILAQCP PGDTVTVGFHDGPNRHTCTVAHKVEFRPVGREKYRHIPPEHGVELPCN RYTHKRADQGHYVEMHQPGLVADHSLLSIHSAKVKITVPSGAQVKYY CKCPDVREGITSSDHTTTCTDVKQCRAYLIDNKKWVYNSGRLPRGEG DTFKGKLHVPFVPVKAKCIATLAPEPLVEHKHRTLILHLHPDHPTLLTT RSLGSDANPTRQWIERPTTVNFTVTGEGLEYTWGNHPPKRVWAQESG EGNPHGWPHEVVVYYYNRYPLTTIIGLCTCVAIIMVSCVTSVWLLCRT RNLCITPYKLAPNAQVPILLALLCCIKPTRADDTLQVLNYLWNNNQNF FWMQTLIPLAALIVCMRMLRCLFCCGPAFLLVCGALGAAAYEHTAVM PNKVGIPYKALVERPGYAPVHLQIQLVNTRIIPSTNLEYITCKYKTKVPS PVVKCCGATQCTSKPHPDYQCQVFTGVYPFMWGGAYCFCDTENTQM SEAYVERSEECSIDHAKAYKVHTGTVQAMVNITYGSVSWRSADVYVN GETPAICIGDAKLIIGPLSSAWSPFDNKVVVYGHEVYNYDFPEYSTGKA GSFGDLQSRTSTSNDLYANTNLKLQRPQAGIVHTPFTQAPSGFERWKR DKGAPLNDVAPFGCSIALEPLRAENCAVGSIPISIDIPDAAFTRISETPTV SDLECKITECTYASDFGGIATVAYKSSKAGNCPIHSPSGVAVIKENDVT LAESGSFTFHFSTANIHPAFKLQVCTSAVTCKGDCKPPKDHIVDYPAQH TESFTSAISATAWSWLKVLVGGTSAFIVLGLIATAVVALVLFFHRH SEQ ID NO: 18 MEKVHVDLDADSPFVKSLQRCFPHFEIEATQVTDNDHANARAFSHLA TKLIEGEVDTDQVILDIGSAPVRHTHSKHKYHCICPMKSAEDPDRLYRY ADKLRKSDVTDKCIASKAADLLTVMSTPDAETPSLCMHTDSTCRYHG SVAVYQDVYAVHAPTSIYYQALKGVRTIYWIGFDTTPFMYKNMAGAY PTYNTNWADESVLEARNIGLGSSDLHEKSFGKVSIMRKKKLQPTNKVI FSVGSTIYTEERILLRSWHLPNVFHLKGKTSFTGRCNTIVSCEGYVVKKI TLSPGIYGKVDNLASTMHREGFLSCKVTDTLRGERVSFPVCTYVPATL CDQMTGILATDVSVDDAQKLLVGLNQRIVVNGRTQRNTNTMQNYLLP VVAQAFSRWAREHRADLEDEKGLGVRERSLVMGCCWAFKTHKITSIY KRPGTQTIKKVPAVFNSFVIPQPTSYGLDIGLRRRIKMLFDAKKAPAPII TEADVAHLKGLQDEAEAVAEAEAVRAALPPLLPEVDKETVEADIDLIM QEAGAGSVETPRRHIKVTTYPGEEMIGSYAVLSPQAVLNSEKLACIHPL AEQVLVMTHKGRAGRYKVEPYHGRVIVPSGTAIPIPDFQALSESATIVF NEREFVNRYLHHIAVNGGALNTDEEYYKVVKSTETDSEYVFDIDAKK CVKKGDAGPMCLVGELVDPPFHEFAYESLKTRPAAPHKVPTIGVYGV PGSGKSGIIKSAVTKRDLVVSAKKENCMEIIKDVKRMRGMDIAARTVD SVLLNGVKHSVDTLYIDEAFACHAGTLLALIAIVKPKKVVLCGDPKQC GFFNMMCLKVHFNHEICTEVYHKSISRRCTKTVTSIVSTLFYDKRMRT VNPCNDKIIIDTTSTTKPLKDDIILTCFRGWVKQLQIDYKNHEIMTAAAS QGLTRKGVYAVRYKVNENPLYAQTSEHVNVLLTRTEKRIVWKTLAG DPWIKTLTASYPGNFTATLEEWQAEHDAIMAKILETPASSDVFQNKVN VCWAKALEPVLATANITLTRSQWETIPAFKDDKAYSPEMALNFFCTRF FGVDIDSGLFSAPTVPLTYTNEHWDNSPGPNMYGLCMRTAKELARRY PCILKAVDTGRVADVRTDTIKDYNPLINVVPLNRRLPHSLVVTHRYTG NGDYSQLVTKMTGKTVLVVGTPMNIPGKRVETLGPSPQCTYKAELDL GIPAALGKYDIIFINVRTPYRHHHYQQCEDHAIHHSMLTRKAVDHLNK GGTCIALGYGTADRATENIISAVARSFRFSRVCQPKCAWENTEVAFVFF GKDNGNHLQDQDRLSVVLNNIYQGSTQHEAGRAPAYRVVRGDITKSN DEVIVNAANNKGQPGGGVCGALYRKWPGAFDKQPVATGKAHLVKHS PNVIHAVGPNFSRLSENEGDQKLSEVYMDIARIINNERFTKVSIPLLSTGI YAGGKDRVMQSLNHLFTAMDTTDADITIYCLDKQWESRIKEAITRKES VEELTEDDRPVDIELVRVHPLSSLAGRPGYSTTEGKVYSYLEGTRFHQT AKDIAEIYAMWPNKQEANEQICLYVLGESMNSIRSKCPVEESEASSPPH TIPCLCNYAMTAERVYRLRMAKNEQFAVCSSFQLPKYRITGVQKIQCS KPVIFSGTVPPAIHPRKFASVTVEDTPMVQPERLVPRRPAPPVPVPARIP SPPCTSTNGSTTSIQSLGEDQSASASSGAEISVDQVSLWSIPSATGFDVR TSSSLSLEQPTFPTMVVEAEIHASQGSLWSIPSITGSETRAPSPPSQDSRP STPSASGSHTSVDLITFDSVAEILEDFSRSPFQFLSEIKPIPAPRTRVTNMS RSADTIKPIPKPRKCQVKYTQPPGVARAISAAEFDEFVRRHSNRRYEAG AYIFSSETGQGHLQQKSTRQCKLQYPILERSVHEKFYAPRLDLEREKLL QKKLQLCASEGNRSRYQSRKVENMKAITVERLLQGIGSYLSAEPQPVE CYKVTYPAPMYSSTASNSFSSAEVAVKVCNLVLQENFPTVASYNITDE YDAYLDMVDGASCCLDTATFCPAKLRSFPKKHSYLRPEIRSAVPSPIQN TLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNDEYWD FYKTNPIRLTAENVTQYVTKLKGPKAAALFAKTHNLQPLHEIPMDRFV MDLKRDVKVTPGTKHTEERPKVQVIQAADPLATAYLCGIHRELVRRL NAVLLPNIHTLFDMSAEDFDAIIAEHFQFGDSVLETDIASFDKSEDDAIA MSALMILEDLGVDQALLNLIEAAFGNITSVHLPTGTRFKFGAMMKSG MFLTLFINTVVNIMIASRVLRERLTTSPCAAFIGDDNIVKGVTSDELMA ERCATWLNMEVKIIDAVVGVKAPYFCGGFIVVDQITGTACRVADPLKR LFKLGKPLPLDDDQDVDRRRALHDEAARWNRIGITEELVKAVESRYE VNYVSLIITALTTLASTVSNFKHIRGHPITLYG SEQ ID NO: 19 AACTAATCGATCCAATATGGAAAGAATTCACGTTGACTTAGATGCT GACAGCCCGTATGTCAAGTCGTTACAGCGGAGCTTTCCACAATTTG AGATCGAAGCAAGGCAGGTCACTGACAATGACCATGCCAATGCCA GAGCGTTTTCGCATGTGGCAACAAAGCTCATTGAGAGCGAAGTCGA CCGGGACCAAGTTATCTTGGACATTGGAAGTGCGCCCGTCAGACAT GCACATTCCAATCACCGCTATCATTGTATCTGCCCTATGATAAGCGC TGAAGACCCGGACAGACTACAACGGTATGCAGAAAGACTTAAGAA AAGTGACATTACCGACAAGAACATAGCCTCTAAGGCGGCAGACCT GCTGGAAGTCATGTCAACACCAGACGCAGAGACTCCATCTCTGTGT ATGCACACAGACGCCACGTGTAGGTACTTTGGAAGTGTAGCAGTAT ACCAAGATGTGTACGCAGTCCATGCACCGACATCAATCTACCACCA GGCGCTTAAAGGAGTTAGGACAATTTACTGGATAGGCTTTGACACG ACCCCTTTTATGTACAAAAACATGGCAGGTTCTTACCCTACTTACAA CACGAACTGGGCTGACGAGAGAGTATTGGAAGCACGTAACATTGG CCTCGGTAACTCAGATCTTCAGGAGAGCAGGCTTGGAAACCTCTCA ATCCTTAGGAAGAAGAGGCTCCAACCTACTAATAAGATCATATTCT CGGTTGGTTCAACAATCTACACAGAAGATAGATCACTGTTACGTAG CTGGCATCTTCCAAACGTGTTCCACTTGAAAGGAAAGTCTAACTTC ACAGGTAGATGTGGGACCATTGTCAGCTGTGAAGGGTACGTCATAA AAAAAATTACAATCAGCCCAGGACTATACGGTAAAGTTGAGAACTT GGCGTCCACAATGCATCGCGAGGGTTTCTTGAGTTGCAAAGTCACA GATACGTTGCGCGGCGAGAGGGTTTCTTTTGCTGTGTGTACGTATGT ACCAGCCACACTTTGCGATCAGATGACAGGGATTCTGGCAACTGAC GTTAGTGTGGATGACGCACAAAAACTATTGGTTGGGCTCAACCAAA GGATTGTCGTCAATGGTAGGACGCAAAGAAATACTAACACAATGC AGAACTATCTATTACCAGTGGTCGCCCAGGCGTTTTCCAGGTGGGC GCGTGAACATCGTGCCGACTTGGACGACGAGAAAGAACTAGGGGT GCGGGAGCGCACTCTTACTATGGGCTGCTGCTGGGCTTTCAAGACC CAGAAAATCACATCCATCTACAAGAAGCCTGGTACGCAAACAATTA AGAAAGTACCTGCCGTCTTTGACTCATTTGTGTTTCCACGCCTTACC AGCCACGGGCTCGATATGGGCTTCCGCCGTAGGCTCAAGCTGCTGC TTGAACCAACTGTCAAACCCGCACCGGCTATTACAATGGCCGATGT GGAGCATCTGCGTGGCTTACAGCAAGAAGCTGAAGAAGTGGCTGC AGCGGAAGAGATCAGAGAAGCCCTGCCGCCCTTGCTCCCTGAAATA GAAAAAGAGACCGTAGAGGCAGAAGTAGACCTCATTATGCAAGAG GCAGGAGCAGGTAGCGTGGAGACACCACGAGGACACATCAGGGTG ACAAGTTACCCAGGCGAAGAGAAGATTGGGTCTTACGCTGTACTTT CACCCCAGGCGGTATTGAATAGTGAAAAACTGGCGTGTATCCACCC ATTGGCGGAACAAGTACTGGTAATGACTCACAAAGGTAGGGCTGG GAGATACAAGGTCGAGCCATACCACGGTAAGGTCATTGTACCAGA AGGGACGGCGGTCCCTGTTCAGGACTTCCAGGCATTGAGTGAGAGC GCTACGATCGTTTTCAACGAGAGGGAGTTCGTAAACAGATACCTGC ATCACATCGCAATCAACGGAGGAGCGCTAAACACTGACGAAGAGT ACTATAAGACTGTAAAGACTCAGGACACAGACTCAGAATACGTCTT CGACGTTGACGCACGAAAGTGTGTTAAGCGAGAAGACGCAGGTCC GTTGTGCCTAACCGGTGATCTGGTAGATCCGCCATTTCACGAGTTTG CGTACGAGAGTCTTAGGACGCGACCAGCAGCACCTCACAAAGTCCC AACCATTGGAGTCTATGGAGTGCCAGGTTCGGGTAAATCTGGAATC ATCAAAAGCGCTGTGACCAAGAAAGATCTGGTTGTGAGTGCGAAG AAGGAAAACTGCGCAGAAATCATCAGGGATGTAAGGAGGATGAGA TGTATGGATGTTGCTGCTAGGACTGTAGATTCTGTGCTGCTGAATGG GGTTAAGCACCCCGTTAACACTCTGTACATTGATGAGGCATTTGCCT GCCATGCAGGGACGCTGCTGGCACTGATTGCCATCGTCAAACCTAA GAAAGTGGTATTGTGCGGGGACCCAAAACAATGCGGCTTCTTTAAC ATGATGTGCCTGAAAGTACATTTTAACCATGACATATGCACTGAAG TGTACCATAAAAGCATCTCTAGGAGGTGCACACAGACTGTAACCGC TATCGTCTCCACGCTCTTTTACGACAAGCGAATGAAGACGGTTAAC CCATGTGCTGACAAAATCATCATAGATACCACAGGGACCACAAAGC CGCACAAAGATGATCTGATTCTAACCTGTTTCAGAGGATGGGTGAA ACAGCTACAGATTGACTATAAAAATCATGAAATCATGACTGCGGCT GCATCGCAAGGACTTACGCGGAAAGGCGTTTATGCTGTCAGGTACA AAGTCAACGAGAATCCACTCTACTCGCAGACTTCTGAGCACGTGAA CGTGTTACTTACACGCACAGAAAAACGCATTGTCTGGAAGACGCTA GCTGGTGATCCCTGGATAAAGATACTTACAGCTAAATATTCCGGGG ATTTCACGGCTTCATTGGACGACTGGCAGCGAGAACATGATGCCAT TATGGCACGCGTTCTTGATAAGCCGCAGACAGCTGATGTGTTCCAG AATAAGGTGAACGTCTGCTGGGCGAAGGCTCTAGAGCCAGTCTTGG CCACGGCCAACATTGTGCTGACGAGACAGCAGTGGGAGACGTTGC ACCCATTCAAGCATGACAGAGCGTACTCACCTGAAATGGCACTGAA CTTCTTTTGCACCAGGTTCTTTGGAGTAGACCTGGACAGTGGGTTGT TTTCCGCTCCTACCGTCGCACTTACTTATAGGGATCAGCACTGGGAT AACTCGCCAGGGAAGAACATGTATGGGCTTAATAGAGAGGTAGCA AAGGAGTTGTCACGGCGATATCCGTGCATCACAAAAGCGGTTGACA CAGGCAGGGTAGCTGATATAAGGAATAATACCATCAAGGACTACTC TCCAACAATTAATGTGGTTCCATTAAATCGTCGGTTACCCCACTCGT TGATCGTTGACCACAAAGGACAGGGTACAACTGATCACAGCGGATT CCTATCTAAGATGAAGGGCAAATCTGTGTTGGTGATCGGCGATCCT ATCAGCATTCCAGGGAAGAAAGTAGAGTCCATGGGTCCATTGCCCA CTAATACCATCAGGTGTGATCTAGATTTGGGAATACCTAGCCATGT CGGTAAATATGACATTATATTTGTCAATGTTAGGACCCCGTATAAG AACCATCACTACCAACAGTGCGAGGATCACGCTATCCACCACAGCA TGTTAACGTGTAAGGCTGTCCACCACCTGAACACTGGCGGAACATG TGTGGCTATAGGGTATGGGCTTGCTGATCGCGCAACCGAGAATATC ATCACTGCGGTAGCGCGCTCATTTAGGTTTACCCGTGTCTGTCAGCC TAAGAACACTGCCGAAAATACTGAGGTTCTCTTCGTGTTCTTCGGC AAGGACAACGGCAACCACACACATGACCAGGACAGACTCGCTGTA GTGCTTGACAACATCTACCAAGGGTCAACCAGGTACGAGGCAGGG AGAGCTCCAGCGTACAGAGTGATCAGAGGTGACATTAGCAAGAGC GCTGACCAAGCTATCGTTAATGCTGCTAATAGCAAAGGTCAACCAG GTTCCGGAGTGTGCGGTGCACTGTACCGAAAATGGCCGGCTGCTTT TGATAGACAGCCAATAGCTGTCGGGACGGCTAGACTTGTGAAGCAC GAACCGCTCATCATACATGCTGTAGGACCCAATTTTTCTAAGATGC CGGAACCGGAGGGCGACCTTAAGCTCGCAGCTGCCTACATGAGCAT AGCGTCAATCGTCAACGCTGAACGGATTACTAAAATATCAGTACCG CTACTGTCAACCGGCATCTATTCTGGTGGCAAAGATCGAGTGATGC AATCATTGCATCACCTGTTCACTGCTTTCGACACTACGGATGCCGAT GTCACCATATATTGCTTGGATAAACAATGGGAGACCAGGATAATCG AGGCCATTCACCGCAAAGAAAGCGTCGAAATACTGGATGATGACA AGCCAGTAGACATTGACTTGGTTAGGGTCCATCCAAACAGCGCTTT GGCAGGCAGACCTGGTTACTCCGTCAATGAGGGCAAGCTGTATTCA TACCTGGAAGGTACACGATTCCATCAGACCGCCAAGGACATTGCCG AAATCCATGCAATGTGGCCCAACAAATCTGAGGCTAATGAGCAGAT TTGCTTGTACATCCTGGGTGAGAGTATGTCCAGCATCCGCTCCAAAT GCCCAGTAGAGGAGTCAGAGGCGTCTGCTCCACCTCACACACTGCC GTGCCTGTGTAATTACGCTATGACGGCTGAGCGCGTATACAGGTTG CGCTCTGCGAAGAAAGAACAGTTCGCCGTATGCTCATCATTCCTGT TGCCGAAGTACAGGATCACAGGCGTGCAGAAGCTACAATGCAGCA AACCAGTCCTGTTTTCAGGCGTCGTACCGCCGGCTGTACACCCCAG GAAGTACGCGGAAATAATTCTAGAAACGCCACCACCGCCAGTAAC GACAACCGTAATATGTGAACCCACTGTGCCAGAACGTATACCCAGT CCGGCGATTTCTAGAGCACCAAGTGCGGAATCACTGCTATCTTTTA GCGGCGTCTCGTTCTCTAGCTCTGCCACACGCTCGTCAACCGCCTGG AGCGACTATGACAGGCGGTTTGTGGTTACAGCTGACGTGCATCAAG CGAACATATCTACGTGGAGCATCCCTAGTGCTCCTGGCTTGGACGT CCAAATACCTTCTGACGTCAGTGATTCCCACTGGAGTGTTCCGAGT GCATCAGGCTTCGAAGTGAGAACACCATCTGTACAGGACCTAACTG CGGAGTGTGCAAAGCCTCGTGGGCTGGCCGAAATAATGCAAGACTT CAATACTGCCCCTTTCCAGTTTCTTTCGGACCACAGACCAGTACCGG CACCACGGAGACGCCCCATCCCATCACCTAGATCGACGGTTTCCGC ACCTCCAGTTCCAAAGCCACGCAGGACTAAGTACCAACAACCACCA GGAGTCGCTAGAGCGATCTCAGAAGCGGAGCTGGACGAGTACATC CGTCAACACTCCAATTGACGGTATGAAGCGGGAGCGTATATTTTCT CATCGGAAACAGGCCAAGGTCACCTTCAACAGAAATCAGTACGTCA ATGTAAACTACAAGAACCTATATTGGACCGGGCCGTCCATGAGAAG TATTACGCCCCGCGCCTCGATCTCGAAAGAGAGAAAATGTTACAGA AGAAATTGCAATTATGTGCCTCTGAAGGAAATAGAAGCAGGTATCA ATCACGAAAAGTAGAAAATATGAAAGCAATTACAGCGGAGCGACT CATTTCTGGATTGGGCACATACCTATCATCAGAAGTGAATCCTGTC GAGTGTTACAGAGTCAACTATCCTGTACCAATCTACTCGTCAACGG TAATTAACAGGTTTACATCTGCAGAGGTCGCGGTTAAAACGTGCAA CTTAGTTATCCAAGAGAATTACCCTACAGTAGCCAGTTATTGTATA ACAGATGAATACGATGCGTATCTTGACATGGTGGACGGCGCATCGT GCTGTCTAGATACAGCCACTTTTTGTCCGGCTAAACTGAGAAGCTA CCCAAAGAAGCATAGCTATTTGCAGCCAGAGATAAGATCAGCCGTC CCATCGCCTATACAGAATACATTACAAAATGTATTGGCTGCAGCTA CTAAAAGGAATTGCAACGTTACCCAAATGCGAGAATTACCTGTCTT AGATTCGGCGGCATTTAACGTTGATTGTTTCAAGAAATACGCATGC AATGATGAGTACTGGGATACCTTTCGCGATAACCCTATTCGGCTAA CTACAGAGAACGTTACGCAATATGTGACAAAGCTGAAAGGGCCGA AAGCAGCAGCATTGTTTGCGAAAACTCACAATCTAAAACCGTTGCA GGAGATACCAATGGATCAATTCGTCATGGATCTAAAAAGAGATGTC AAAGTTACTCCCGGCACGAAACATACAGAGGAGCGGCCTAAGGTG CAGGTTATTCAGGCTGCAGACCCCCTTGCTACCGCTTACCTTTGCGG GATCCACCGGGAATTAGTCCGTAGACTGAACGCTGTGCTTCTGCCG AATATCCATACTCTCTTCGACATGTCAGCGGAAGATTTTGATGCGAT TATTGCTGGACATTTCCACCACGGCGACCCAGTATTGGAAACGGAC ATCGCGTCGTTTGATAAAAGCGAAGACGACGCTATCGCCATTTCGG CGATGATGATCCTTGAGGACTTAGGCGTCGACCAACCGCTCTTAGA TTTGATAGAGGCGGCGTTCGGCAATATCACATCTGTGCACCTACCT ACAGGAACGAGGTTTAAATTTGGTGCCATGATGAAATCCGGCATGT TCTTAACGCTGTTTGTCAACACACTAGTCAATATCATGATTGCTAGC AGAGTACTACGTGAACGGTTAACCACGTCAGCGTGCGCGGCCTTTA TCGGCGACGATAACATAGTGCATGGTGTCGTCTCCGACACCTTGAT GGCGGAGAGATGCGCCACTTGGCTGAACATGGAAGTAAAAATTATT GATGCAGTCATTGGTATCAAAGCACCCTACTTCTGCGGGGGATTTA TCCTGGTGGACCAGATAACAGGCACAGCCTGCAGGGTCGCAGACCC TCTAAAAAGGCTTTTTAAGCTTGGAAAACCGTTGTCAGTTGAAGAC ACCCAAGACTGCGACCGCCGCCGGGCACTGCATGATGAAGCAATG CGATGGAACAGAATCGGAATTACGGACGAGTTGGTGAAGGCCGTA GAATCCAGATACGAGATCATACTGGCAGGCCTGATCATCACGTCTC TGTCCACGTTAGCCGAAAGCGTTAAGAACTTCAAGAGCATAAGAGG GAGCCCAATCACCCTCTACGGCTGACCTAAATAGGTGACGTAGTAG ACACGCACCTACCCATCGCCATAATGTTTCCATACCCTCAGCTGAA CTTTCCACCAGTTTACCCTACAAATCCGATGGCTTACCGAGATCCAA ACCCTCCTAGGCGCCGCTGGAGGCCGTTCCGGCCCCCGCTGGCTGC TCAAATCGAAGATCTTAGGAGGTCGATAGCTAACTTGACTTTCAAA CAACGATCACCTAATCCGCCGCCAGGTCCACCGCCAAAGAAGAAG AAGAGTGCTCCTAAGCCAAAACCTACTCAGCCTAAAAAGAAGAAG CAGCAAGCCAAGAGGACGAAACGTAAGCCTAAACCAGGGAAACGA CAGCGTATGTGTATGAAGTTGGAGTCGGACAAGACATTTCCGATCA TGCTGAACGGCCAAGTGAATGGATACGCTTGCGTTGTCGGAGGAAG GCTGATGAAACCACTCCACGTTGAAGGAAAAATTGATAATGAGCA ATTAGCGGCCGTGAAATTGAAGAAGGCTAGCAAGTACGACTTAGA GTATGGCGACGTTCCCCAGAATATGAAATCAGACACGCTGCAGTAC ACCAGTGACAAACCACCGGGCTTCTACAACTGGCACCATGGCGCAG TCCAGTATGAGAATGGGAGATTCACCGTACCGAGAGGAGTGGGCG GGAAAGGCGACAGCGGAAGACCGATCCTGGACAACAGAGGCAGAG TTGTGGCTATTGTTCTAGGAGGTGCAAATGAGGGCACGCGTACGGC GCTTTCAGTGGTCACTTGGAACCAGAAAGGGGTGACCGTTTGGGAT CCCCCCGAAGGTTCTGAACCGTGGTCACTAGTTACAGCGCTGTGCG TGCTTTCGAATGTCACTTTCCCATGTGACAAACCACCCGTGTGCTAT TCACTGGCGCCAGAACGAACACTCGACGTGCTCGAAGAGAACGTC GACAATCCAAATTACGACACGCTGCTGGAGAACGTCTTGAAATGTC CATCACACCGGCCCAAACGAAGCATTACCGATGACTTCACACTGAC CAGTCCCTACTTGGGGTTCTGCCCGTATTGCAGACACTCAACGCCGT GTTTCAGCCCAATAAAAATTGAGAACGTGTGGGACGAATCTGATGA TGGATCGATTAGAATCCAGGTCTCGGCACAATTCGGTTACAATCAG GCAGGCACTGCAGATGTCACTAAATTCCGTTACATGTCTTTCGACC ACGACCATGACATCAAGGAAGACAGTATGGAGAAAATAGCCATCA GCACATCCGGACCCTGCCGTCGTCTTGGCCACAAAGGGTATTTCCT GTTAGCTCAATGTCCTCCAGGTGACAGTGTAACCGTCAGTATCACG AGCGGAACTGCTGAGAACTCATGCACCGTGGAGAGAAAGATCAGG AGGAAGTTTGTCGGTAGAGAGGAGTACTTGCTCCCACCCATCCATG GAAAGCAGGTAAAGTGCCACGTTTACGATCACTTGAAAGAGACGTC TGCCGGGTATATAACCATGCACAGGCCAGGCCCACACGCGTATAAG TCCTATCTGGAGGAAGCGTCAGGCGAAGTGTATATTAAACCACCTT CTGGCAAGAACGTCACCTACGAATGTAAGTGTGGCGACTACAGCAC AGGTATCGTGAGCACGCGAACGAAGATAAACGGCTGCACTAAAGC AAAACAGTGCATTGCCTACAAGAGCGACCAAACGAAATGGGTCTTC AACTCGCCGGATCTTATTAGGCACACAGACCACTCAGTGCAAGGTA AACTGCATATTCCATTCCGCTTGACACCGACAGTCTGCCCGGTTCCG TTAGCTCACACGCCTACAGTCATGAAGTGGTTCAAAGGCATCACCC TCCACCTGACTGCAACGCGACCAACATTGCTGACAACGAGAAAATT GGGGCTGCGAGCAGACGCAACAGCAGAATGGATTACAGGGACTAC ATCCAGGAATTTTTCTGTGGGGCGAGAAGGGCTGGAGTACGTATGG GGCAACCATGAACCGGTCAGAGTCTGGGCCCAGGAGTCGGCACCA GGCGACCCACATGGATGGCCGCATGAGATTATCATTCACTATTATC ATCGGCATCCAGTCTACACCGTCATTGTGCTGTGTGGTGTCGCTCTT GCTATCCTGGTAGGCACTGCATCGTCAGCAGCTTGTATCTCCAAAG CAAGAAGAGACTGCCTGACGCCATACGCGCTTGCACCGAACGCGA CGGTACCCACAGCATTAGTGGTTTTGTGCTGCATTCGGCCAACCAA CGCTGAAACACTTGGAGAAACTTTGAACCATCTGTGGTTTAACAAC CAACCGTTTCTCTGGGCACAGTTGTGCATTCCTCTGGCGGCTCTTAT TATTCTGTTCCGCTGCTTTTCATGCTGCATGCCTTTTTTATTGGTTGC AGGCGTCTGCCTGGGGAAGGTAGACGCCTTCGAACATGCGACCACT GTGCCAAATGTTCCGGGGATCCCGTATAAGGCGTTGGTCGAACGTG CAGGTTACGCGCCACTTAACCTGGAGATCACTGTTGTCTCATCGGA ATTAACACCCTCAACTAATAAGGAGTACGTGACCTGCAAATTCCAC ACAGTCATTCCTTCACCACAAGTTAAATGCTGCGGGTCCCTCGAGT GTAAGGCATCCTCAAGGGCGGATTACACATGCCGCGTTTTTGGCGG TGTGTACCCTTTCATGTGGGGAGGCGCACAATGCTTCTGTGACAGT GAGAACACACAACTGAGTGAGGCATACGTCGAGTTCGCTCCGGACT GCACTATAGATCACGCAGTCGCACTAAAAGTTCATACAGCTGCTCT GAAAGTCGGCCTGCGTATAGTGTACGGTAATACCACCGCGCACCTG GATACGTTCGTCAACGGCGTCACACCAGGTTCCTCACGGGACCTGA AGGTCATAGCAGGGCCGATATCAGCCGCTTTTTCACCCTTTGACCAT AAGGTCGTCATCAGAAAGGGGTTTGTTTACAACTACGACTTCCCTG AGTATGGTGCTATGAAACCAGGAGCGTTCGGCGATATTCAAGCATC CTCTCTTGATGCTACAGACATAGTAGCCCGCACTGACATACGGCTG CTGAAGCCTTCTGTCAAGAACATCCACGTCCCCTACACCCAAGCAG TATCAGGGTATGAAATGTGGAAGAACAACTCAGGACGACCCCTGC AAGAAACAGCACCATTTGGATGTAAAATTGAAGTGGAGCCTCTGCG AGCGTCTAACTGTGCTTACGGGCACATTCCTATCTCGATTGACATCC CTGATGCAGCTTTCGTGAGATCATCAGAATCACCAACAATTTTAGA AGTTAGCTGCACAGTAGCAGACTGCATTTATTCTGCAGACTTTGGT GGTTCTCTAACATTACAGTACAAAGCTGATAGGGAGGGACATTGTC CAGTTCACTCCCACTCCACGACAGCTGTTTTGAAGGAAGCGACCAC ACATGTGACTGCCGTAGGCAGCATAACACTACATTTTAGCACATCG AGCCCACAAGCAAATTTTATAGTTTCGCTATGCGGCAAGAAGTCCA CCTGCAATGCTGAATGTAAACCACCGGCCGACCACATAATTGGAGA ACCGCATAAAGTCGACCAAGAATTCCAAGCGGCAGTTTCCACAACA TCTTGGAACTGGCTGCTTGCACTGTTTGGGGGAGCATCATCCCTCAT TGTTGTAGGACTTATAGTGTTGGTCTGCAGCTCTATGCTTATAAACA CACGTAGATGACTGAGCGCGGACACTGACATAGCGGTAAAAACTC GATGTACTTCCGAGGAAGCGTGGTGCATAATGCCACGCGCCGCTTG ACACTAAAACTCGATGTATTTCCGAGGAAGCACAGTGCATAATGCT GTGCAGTGTCACATTAATCGCATATCACACTATATATTAACAACAC TATATCACTTTTATAAGACTCACTATGGGTCTCTAATATACACTACA CATATTTTACTTAAAAACACTATACACACTTTATAAGTTCTTTTATA ATTTTTCTTTTGTTTTTATTTTGTTTTTAAAATTT SEQ ID NO: 20 MFPYPQLNFPPVYPTNPMAYRDPNPPRRRWRPFRPPLAAQIEDLRRSIA NLTFKQRSPNPPPGPPPKKKKSAPKPKPTQPKKKKQQAKRTKRKPKPG KRQRMCMKLESDKTFPIMLNGQVNGYACVVGGRLMKPLHVEGKIDN EQLAAVKLKKASKYDLEYGDVPQNMKSDTLQYTSDKPPGFYNWHHG AVQYENGRFTVPRGVGGKGDSGRPILDNRGRVVAIVLGGANEGTRTA LSVVTWNQKGVTVWDPPEGSEPWSLVTALCVLSNVTFPCDKPPVCYS LAPERTLDVLEENVDNPNYDTLLENVLKCPSHRPKRSITDDFTLTSPYL GFCPYCRHSTPCFSPIKIENVWDESDDGSTRIQVSAQFGYNQAGTADVT KFRYMSFDHDHDIKEDSMEKIAISTSGPCRRLGHKGYFLLAQCPPGDS VTVSITSGTAENSCTVERKIRRKFVGREEYLLPPIHGKQVKCHVYDHLK ETSAGYITMHRPGPHAYKSYLEEASGEVYTKPPSGKNVTYECKCGDYS TGIVSTRTKINGCTKAKQCIAYKSDQTKWVFNSPDLIRHTDHSVQGKL HIPFRLTPTVCPVPLAHTPTVMKWFKGITLHLTATRPTLLTTRKLGLRA DATAEWITGTTSRNFSVGREGLEYVWGNHEPVRVWAQESAPGDPHG WPHEIIIHYYHRHPVYTVIVLCGVALAILVGTASSAACISKARRDCLTP YALAPNATVPTALVVLCCIRPTNAETLGETLNHLWFNNQPFLWAQLCI PLAALIILFRCFSCCMPFLLVAGVCLGKVDAFEHATTVPNVPGIPYKAL VERAGYAPLNLEITVVSSELTPSTNKEYVTCKFHTVIPSPQVKCCGSLE CKASSRADYTCRVFGGVYPFMWGGAQCFCDSENTQLSEAYVEFAPDC TIDHAVALKVHTAALKVGLRIVYGNTTAHLDTFVNGVTPGSSRDLKVI AGPISAAFSPFDHKVVIRKGFVYNYDFPEYGAM1CPGAFGDIQASSLDA TDIVARTDIRLLKPSVKNIHVPYTQAVSGYEMWKNNSGRPLQETAPFG CKIEVEPLRASNCAYGHIPISIDIPDAAFVRSSESPTILEVSCTVADCIYSA DFGGSTITQYKADREGHCPVHSHSTTAVLKEATTHVTAVGSITLHTST SSPQANFIVSLCGKKSTCNAECKPPADHIIGEPHKVDQEFQAAVSTTSW NWLLALFGGASSLIVVGLIVLVCSSMLINTRR SEQ ID NO: 21 MERIHVDLDADSPYVKSLQRSFPQFETEARQVIDNDHANARAFSHVAT KLIESEVDRDQVILDIGSAPVRHAHSNHRYHCICPMISAEDPDRLQRYA ERLKKSDITDKNIASKAADLLEVMSTPDAETPSLCMHTDATCRYFGSV AVYQDVYAVHAPTSIYHQALKGVRTIYWIGFDTTPFMYKNMAGSYPT YNTNWADERVLEARNIGLGNSDLQESRLGNLSILRKKRLQPTNKTIFSV GSTIYTEDRSLLRSWHLPNVFHLKGKSNFTGRCGTIVSCEGYVIKKITIS PGLYGKVENLASTMHREGFLSCKVTDTLRGERVSFAVCTYVPATLCD QMIGILATDVSVDDAQKLLVGLNQRIVVNGRTQRNTNTMQNYLLPV VAQAFSRWAREHRADLDDEKELGVRERTLTMGCCWAFKTQKITSTYK KPGTQTIKKVPAVFDSFVFPRLTSHGLDMGFRRRLKLLLEPTVKPAPAI TMADVEHLRGLQQEAEEVAAAEEIREALPPLLPEIEKETVEAEVDLIMQ EAGAGSVETPRGHIRVTSYPGEEKIGSYAVLSPQAVLNSEKLACIHPLA EQVLVMTHKGRAGRYKVEPYHGKVIVPEGTAVPVQDFQALSESATIV FNEREFVNRYLHHIAINGGALNTDEEYYKTVKTQDTDSEYVFDVDAR KCVKREDAGPLCLTGDLVDPPFHEFAYESLRTRPAAPHKVPTIGVYGV PGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVRRMRCMDVAARTVD SVLLNGVICHPVNTLYIDEAFACHAGTLLALIAIVKPKKVVLCGDPKQC GFFNMMCLKVHFNHDICTEVYHKSISRRCTQTVTAIVSTLFYDKRMKT VNPCADKIIIDTTGTTKPHKDDLILTCFRGWVKQLQIDYKNHEIMTAAA SQGLTRKGVYAVRYKVNENPLYSQTSEHVNVLLTRTEKRIVWKTLAG DPWIKILTAKYSGDFTASLDDWQREHDAIMARVLDKPQTADVFQNKV NVCWAKALEPVLATANIVLTRQQWETLHPFKHDRAYSPEMALNFFCT RFFGVDLDSGLFSAPTVALTYRDQHWDNSPGKNMYGLNREVAKELSR RYPCITKAVDTGRVADIRNNTIKDYSPTINVVPLNRRLPHSLIVDHKGQ GTTDHSGFLSKMKGKSVLVIGDPISIPGKKVESMGPLPTNTIRCDLDLGI PSHVGKYDIIFVNVRTPYKNHHYQQCEDHAIHHSMLTCKAVHHLNTG GTCVAIGYGLADRATENIITAVARSFRFTRVCQPKNTAENTEVLFVFFG KDNGNHTHDQDRLAVVLDNIYQGSTRYEAGRAPAYRVIRGDISKSAD QAIVNAANSKGQPGSGVCGALYRKWPAAFDRQPIAVGTARLVKHEPL IIHAVGPNFSKMPEPEGDLKLAAAYMSIASIVNAERITKISVPLLSTGIYS GGKDRVMQSLHHLFTAFDTTDADVTIYCLDKQWETRIIEAIHRKESVEI LDDDKPVDIDLVRVHPNSALAGRPGYSVNEGKLYSYLEGTRFHQTAK DIAEIHAMWPNKSEANEQICLYILGESMSSIRSKCPVEESEASAPPHTLP CLCNYAMTAERVYRLRSAKKEQFAVCSSFLLPKYRITGVQKLQCSKPV LFSGVVPPAVHPRKYAEIILETPPPPVTTTVICEPTVPERIPSPAISRAPSA ESLLSFSGVSFSSSATRSSTAWSDYDRRFVVTADVHQANISTWSIPSAP GLDVQIPSDVSDSHWSVPSASGFEVRTPSVQDLTAECAKPRGLAEIMQ DFNTAPFQFLSDHRPVPAPRRRPIPSPRSTVSAPPVPKPRRTKYQQPPGV ARAISEAELDEYIRQHSNRYEAGAYIFSSETGQGHLQQKSVRQCKLQEP ILDRAVHEKYYAPRLDLEREKMLQKKLQLCASEGNRSRYQSRKVENM KAITAERLISGLGTYLSSEVNPVECYRVNYPVPIYSSTVINRFTSAEVAV KTCNLVIQENYPTVASYCITDEYDAYLDMVDGASCCLDTATFCPAKLR SYPKKHSYLQPEIRSAVPSPIQNTLQNVLAAATKRNCNVTQMRELPVL DSAAFNVDCFKKYACNDEYWDTFRDNPIRLTTENVTQYVTKLKGPKA AALFAKTHNLKPLQEIPMDQFVMDLKRDVKVTPGTKHTEERPKVQVI QAADPLATAYLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAGHF HHGDPVLETDIASFDKSEDDAIAISAMMILEDLGVDQPLLDLIEAAFGNI TSVHLPTGTRFKFGAMMKSGMFLTLFVNTLVNIMIASRVLRERLTTSA CAAFIGDDNIVHGVVSDTLMAERCATWLNMEVKIIDAVIGIKAPYFCG GFILVDQITGTACRVADPLKRLFKLGKPLSVEDTQDCDRRRALHDEAM RWNRIGITDELVKAVESRYEIILAGLIITSLSTLAESVKNFKSIRGSPITLY G SEQ ID NO: 48 MEGDGSDPEPPDAGEDSKSENGENAPIYCICRKPDINCFMIGCDNCNE WFHGDCIRITEKMAKAIREWYCRECREKDPKLEIRYRHKKSRERDGNE RDSSEPRDEGGGRKRPVPDPNLQRRAGSGTGVGAMLARGSASPHKSS PQPLVATPSQHHQQQQQQIKRSARMCGECEACRRTEDCGHCDFCRDM KKFGGPNKIRQKCRLRQCQLRARESYKYFPSSLSPVTPSESLPRPRRPLP TQQQPQPSQKLGRIREDEGAVASSTVKEPPEATATPEPLSDEDLPLDPD LYQDFCAGAFDDNGLPWMSDTEESPFLDPALRKRAVKVKHVKRREK KSEKKKEERYKRHRQKQKHKDKWKHPERADAKDPASLPQCLGPGCV RPAQPSSKYCSDDCGMKLAANRIYEILPQRIQQWQQSPCIAEEHGKKL LERIRREQQSARTRLQEMERRFHELEAHLRAKQQAVREDEESNEGDSD DTDLQIFCVSCGHPINPRVALRHMERCYAKYESQTSFGSMYPTRIEGAT RLFCDVYNPQSKTYCKRLQVLCPEHSRDPKVPADEVCGCPLVRDVFEL TGDFCRLPKRQCNRHYCWEKLRRAEVDLERVRVWYKLDELFEQERN VRTAMTNRAGLLALMLHQTIQHDPLTTDLRSSADR 

1. A composition comprising a replication defective virus vector comprising a sequence encoding an alphavirus target antigen.
 2. The composition of claim 1, wherein the sequence encoding the alphavirus target antigen comprises a sequence encoding a plurality of alphavirus target antigens.
 3. The composition of claim 2, wherein the sequence encoding a plurality of alphavirus target antigens comprises a plurality of gene inserts each corresponding to a target antigen and wherein each gene insert is separated by a nucleic acid sequence encoding a self-cleaving 2A peptide.
 4. The composition of claim 3, wherein the self-cleaving 2A peptide is derived from Porcine teschovirus-1 or Thosea asigna virus.
 5. The composition of claim 1, wherein the replication defective virus vector is an adenovirus vector.
 6. (canceled)
 7. The composition of claim 1, wherein the replication defective virus vector comprises an adenovirus vector with a deletion in an E1 gene region, an E2b gene region, an E3 gene region, E4 gene region, or any combination thereof.
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. The composition of claim 1, wherein the alphavirus target antigen comprises an antigen of a virus selected from the group consisting of Chikungunya virus (CHIKV), o'nyong-nyong virus (ONNV), Ross River virus (RRV), Mayaro fever virus (MAYV), Venezuelan equine encephalitis virus (VEEV), Western equine encephalomyelitis virus (WEEV), and Eastern equine encephalitis virus (EEEV), or any combination thereof.
 14. (canceled)
 15. (canceled)
 16. The composition of claim 1, wherein the alphavirus target antigen comprises an antigen selected from the group consisting of C, E3_(ALPHA), E2_(ALPHA), 6K, E1_(ALPHA), nsP1, nsP2, nsP3, and nsP4, or any combination thereof.
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. The composition of claim 1, wherein the replication defective virus vector further comprises an element to increase an expression of the alphavirus target antigen.
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. The composition of claim 1, wherein the replication defective virus vector further comprises a nucleic acid sequence encoding a protein that increases alphavirus target antigen immunogenicity.
 32. (canceled)
 33. The composition of claim 1, wherein the composition or the replication defective virus vector further comprises a sequence encoding a costimulatory molecule or an immunological fusion partner.
 34. (canceled)
 35. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
 36. A cell comprising the composition according to the pharmaceutical composition of claim
 35. 37. (canceled)
 38. The cell of claim 36, wherein the cell is a dendritic cell (DC).
 39. A method of preparing a vaccine, comprising preparing a composition of claim
 1. 40. A method of generating an immune response against an alphavirus target antigen in a subject, comprising: administering to the subject a composition according to claim
 1. 41. (canceled)
 42. (canceled)
 43. A method of preventing a Chikungunya virus infection in a subject, the method comprising administering to the subject a composition comprising: a replication defective virus vector comprising a deletion in an E2b gene region; and a nucleic acid sequence encoding at least one Chikungunya target antigen.
 44. The method of claim 43, wherein the subject has preexisting immunity to an adenovirus or an adenovirus vector.
 45. (canceled)
 46. (canceled)
 47. (canceled)
 48. The method of claim 43, wherein the administering to the subject comprises 1×10⁹ to 5×10¹² virus particles per dose.
 49. (canceled)
 50. The method of claim 43, wherein the replication defective virus vector is an adenovirus vector.
 51. (canceled) 